Caracterización de aislamientos de Klebsiella pneumoniae doble productores de carbapenemasas en un hospital universitario / Phenotypic and genotypic characterization of double carbapenemase producing Klebsiella pneumoniaeisolates in a University Hospital

La emergencia de aislamientos de Klebsiella pneumoniaedoble productores de carbapenemasas (KPC y NDM) es una de las consecuencias de la pandemia causada por SARS-CoV-2 que ha causado un impacto significativo en las tasas de resistencia a los antimicrobianos en las infecciones intrahospitalarias por esta enterobacteria. Estos aislamientos representan un desafío para los servicios de salud, por su detección y caracterización y posterior tratamiento. En este trabajo se describen los aislamientos portadores de KPC y NDM recuperados durante 2022 aislados de distintas muestras clínicas de pacientes internados en un hospital universitario de la Ciudad de Buenos Aires, se los caracteriza fenotípicamente y genotípicamente como portadores de ambas carbapenemasas y se destaca la excelente actividad in vitro de la combinación ceftazidima-avibactam y aztreonam en el tratamiento de estas infecciones en donde las alternativas terapéuticas estarían limitadas a antibióticos no ß-lactámicos con porcentajes de resistencia que superan el 70%

The emergence of double-carbapenemase (KPC and NDM) producing Klebsiella pneumoniae isolates is one of the consequences derived from the SARS CoV-2 pandemic, which has caused significant impact on the antimicrobial resistance rates in hospital acquired infections. These isolates represent a real challenge for Health Services due to their difficult detection and characterization and subsequent treatment. In the present work we describe the double carbapenemase producing isolates recovered during the year 2022 from clinical samples belonging to hospitalized patients at a University Hospital in Buenos Aires city, we report their phenotypic and genotypic characterization and the excellent "in vitro" activity of the ceftazidime-avibactam-aztreonam combination in the treatment of infections in which the therapeutical options are restricted to non ß- lactamic antimicrobials which hold resistance rates higher than 70%

Emergence and clonal expansión of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1

Abstract MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describesthe emergence and clonal dissemination of K. pneumoniae ST307, recovered during November2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3and NDM-1. These isolates were resistant to all -lactams and to the ceftazidime/avibactamcombination. Molecular studies showed that blaKPC-3was located in Tn4401a platform, whileblaNDM-1was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 anddownstream by bleMBL-trpF, located in a 145.5 kb conjugative plasmid belonging to the Inc A/Cgroup. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 couldlead to a worrisome scenario due to the remarkable features of this clone and its resistanceprofile.

Resumen Klebsiella pneumoniae ST307 es un clon de alto riesgo, cuyas características genéticas contribuyen a su adaptación al entorno hospitalario y al huésped humano. Este estudio describe la emergencia y diseminación clonal de aislamientos de K. pneumoniae ST307 productores de KPC-3 y NDM-1, recuperados en un hospital de Buenos Aires. Estos aislamientos fueron resistentes a todos los p-lactámicos y a la combinación ceftacidima/avibactam. Los estudios moleculares evidenciaron que el contexto genético de blaKPC-3 se correspondió con el Tn4401a, mientras que blaNDM-1 estuvo flanqueado corriente arriba por ISKpn14 y una secuencia parcial de ISAba125 y corriente abajo por bleMBL - trpF, localizado a su vez en un plásmido conjugativo de 145.5 kb perteneciente al grupo Inc A/C. La emergencia de aislamientos de K. pneumoniae ST307 coproductores de KPC-3 y NDM-1 pone de manifiesto una situación altamente preocupante debido a las características de este clon y a su perfil de multirresistencia.

DETECTION OF CARBAPENEMASE PRODUCING ENTEROBACTERALES AMONG THE CLINICAL ISOLATES OF DIARRHOEAGENIC ESCHERICHIA COLI CAUSING ACUTE GASTROENTERITIS IN CHILDREN BELOW 2 YEARS IN TERTIARY CARE HOSPITAL OF NORTH EAST INDIA.

Introduction: Enterobacterales that test resistant to at least one of the carbapenem antibiotics (ertapenem, meropenem, doripenem, or imipenem) are called Carbapenem resistant Enterobacterales (CRE) and if they produce a carbapenemase (an enzyme that can make them resistant to carbapenem antibiotics) they are called Carpenemase producing Enterobacterales (CPE). Children with CRE strains in fecal samples are considered as a high risk group by World Health Organization (WHO), which can spread CRE by intimate contact and travel. This cross-sectional study was conducted in the Departm Methods: ent of Microbiology, RIMS, Imphal, Manipur, India from Jan 2020 to Feb 2022. A total of 157 children under 2 years of age whose stool culture was positive for diarrhoeagenic Escherichia coli were included in the study. The modified carbapenem inactivation method (mCIM) has been done for detection of carbapenemase producers and the addition of EDTA in eCIM to further differentiate between serine and metallo-?-lactamase producers. Out of 157 Result and Discussion: Diarrhoegenic E.coli (DEC) ,Carbapenem resistance was seen in 9 isolates i.e 5.7 %. Out of these 9 isolates, 3 were MBL producers tested by the phenotypic test mCIM and eCIM. All the three MBL producers carried bla NDM-1 gene. mCIM/eCIM assay is designed to simultaneously detect and distinguish the different types of carbapenemases. Carbapenemase genes are often located on plasmids that can be exchanged between Enterobacteriaceae and other Gram-negative bacteria. Carbapenem-resistant K. pneumoniae are currently more frequent and more likely to cause healthcareassociated outbreaks, carbapenem-resistant E. coli pose a greater risk for spread in the community. Conclusion: Screening for carbapenemase producer using mCIM and eCIM essay is important along with infection control measure such as active surveillance through rectal screening for CRE carriage on hospital admission, contact precautions, hand hygiene, patient isolation, environmental sanitation, case notification/fiagging, antibiotic restriction.

Primeros aislados de Enterobacter cloacae complex co-productores de KPC y NDM en un hospital de segundo nivel en la Cuidad de Panamá / First isolates of Enterobacter cloacae complex co-producing KPC and NDM in a second level hospital in City of Panama

Resumen La aparición de Enterobacterales co-productores de dos o más carbapenemasas han despertado las alertas sanitarias en Latinoamérica. Las enterobacterias co-productoras de carbapenemasas KPC y NDM-1 son resistentes a casi todos los antibacterianos existentes. Panamá ha reportado la presencia de carbapenemasas KPC desde 2010 y NDM desde 2011; sin embargo, Enterobacterales con doble producción de carbapenemasas es un fenómeno reciente en nuestros hospitales. Presentamos los dos primeros aislados de Enterobacter cloacae complex co-productores de KPC y NDM, en un hospital de segundo nivel de la Ciudad de Panamá. El reforzamiento de los sistemas de vigilancia epidemiológica en los hospitales permite realizar una detección oportuna de estas nuevas combinaciones de mecanismos de resistencia; para así, implementar medidas de prevención y control de brotes.

Abstract Enterobacterales co-producing carbapenemases have awakened health alerts in Latin America. Carbapenemase-producing Enterobacterales harboring KPC and NDM-1 are resistant to almost all existing antibiotics. Panama reports KPC since 2010, and NDM since 2011, however, Enterobacterales with double carbapenemase production is new to our hospitals. We present the first two isolates of Enterobacter cloacae complex co-producing KPC and NDM, in a second level hospital in Panama City. Strengthening epidemiological surveillance systems in hospitals allows to carry out timely detection of these new combinations of resistance; to implement outbreak prevention and control measures.

Susceptibility testing for aztreonam plus ceftazidime/avibactam combination: A general guidance for clinical microbiology laboratories in India

Metallo beta-lactamases-producing Gram-negative infection is often challenging and there is no defined treatment option. In recent years, the combination of aztreonam with ceftazidime-avibactam has gained much clinical attention mainly for MBL-producing Enterobacterales, while MBL-producing P. aeruginosa and A. baumannii are likely to be resistant. A consensus susceptibility testing method for this triple combination has yet to be recommended. Various methods such as broth disk elution, disk stacking, gradient strip stacking, and strip crossing have been proposed for testing this combination. Among them, broth disk elution and strip based testing methods showed good correlation with the broth micro-dilution method.

NDM-beta-lactamase-1: Where do we stand?

Multidrug-resistant (MDR) Gram-negative bacilli (GNB) have been playing havoc in the field of nosocomial as well as community-acquired infections. Of particular concern are the carbapenem-resistant GNBs, belonging to Enterobacteriaceae and encoding for New Delhi metallo-beta-lactamase-1 (NDM-1) gene. These strains spread rapidly and horizontally in the population, thus exhibiting MDR traits as these can harbour several resistance encoding genes to almost all antimicrobial groups. Several predisposing factors are responsible towards its spread, viz. excessive antibiotic usage, improper aseptic conditions by healthcare workers, lack of awareness, abruptly discontinuing medication course, alternative medications and vector-borne factors contributing to the unchecked harbouring of these super bugs in India. Thus, a bugle call has already been sounded worldwide especially in India, where the country has taken serious cognizance to build up strategy via implementation of several national programs to combat antimicrobial resistance covering human, animal, agriculture and environmental aspects. As there is an exponential rise in variants of NDM-1 harbouring strains, molecular epidemiological investigations of these strains using genotyping techniques are of paramount importance for a better understanding of this rampant spread and curbing resistance thereafter. This review explores the urgent need to develop a cost-effective, rapid molecular assay, viz. the loop-mediated isothermal amplification method for field detection of MBL harbouring bacterial strains, especially NDM-1 and its variants, thus targeting specific carbapenemase genes at a grass root level even to the remote and rural regions of the country.

Genomic characteristics of a Klebsiella pneumoniae strain coproducing carbapenemases KPC-2 and NDM-5 / 中华微生物学和免疫学杂志

Objective:To analyze the characteristics of drug resistance genes in a Klebsiella pneumoniae strain coproducing carbapenemases KPC-2 and NDM-5. Methods:Klebsiella pneumoniae KPN-hnqyy was separated from the stool specimen of a patient in the Hematology Department of Affiliated Cancer Hospital of Zhengzhou University. The strain was identified with a BD Phenix-M50 automated microbiology system and the minimum inhibitory concentration against the strain was measured as well. The genotypes of the carbapenemases were tested by enzyme immunochromatographic assay and PCR method. The transferability of related plasmids was analyzed by conjugation test. Whole-genome sequencing of the strain was conducted using PacBio and Illumina platforms. The MLST type, resistance gene and plasmid type of the strain were retrieved in BacWGSTdb. The genome and open reading frame sequence of the strain were compared using Easyfig_2.2.3. Visual cycle graphs were generated using BRIG v0.95. Results:Klebsiella pneumoniae KPN-hnqyy was resistant to carbapenem antibiotics. It belonged to ST11 and carried two carbapenemase genes of blaKPC-2 and blaNDM-5. The conjugant only harbored the blaKPC-2 gene. Whole-genome sequencing revealed that the strain contained one chromosome and three plasmids. Its chromosome genome shared more than 99.9% similarity with that of Klebsiella pneumonia KP69 and KP19-2029. Moreover, a similar IncR and IncFⅠ resistance gene fusion region was contained in different types of plasmids carried by them: the blaKPC-2 gene was located in a structure—which evolved from the Tn3-△Tn4401-Tn1721/Tn1722 sequence—inside this fusion region with its ends inserted into the transposase IS26 gene; the blaNDM-5 gene was located on a transposon containing the special plasmids of the insertion fragment in phages, with its ends inserted into the transposase IS26 gene too. Conclusions:The IncR and IncFⅡ resistance gene fusion region of blaKPC-2 carried by Klebsiella pneumoniae ST11 might be widely coexistent with the chromosomal genome. The blaNDM-5 gene carried by special plasmids might be accidentally obtained through gene recombination mediated by transposable element IS26. The wide transmission of Klebsiella pneumoniae ST11 carrying the blaKPC-2 gene in China and its ability to obtain other carbapenemase genes through transposable element IS26 were well worth attention.

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

Primer reporte de Enterobacterales dobles productores de carbapenemasas en hospitales de Paraguay. Año 2021

RESUMEN Las carbapenemasas se encuentran ampliamente distribuidas en nuestro país, tanto en bacilos gramnegativos fermentadores como no fermentadores. Durante 2021, se ha reportado incremento de cepas con estas enzimas. Con el objetivo de evaluar la doble producción de carbapenemasas en Enterobacterales y comunicar su circulación, fue puesta a punto una PCR convencional múltiple. Estudio retrospectivo en 128 aislamientos provenientes de 20 centros colaboradores de la Red Nacional de Vigilancia de la RAM (Capital, Central e interior del país), remitidos al LCSP entre febrero y setiembre de 2021, para confirmación y genotipificación de carbapenemasas. Se realizaron pruebas fenotípicas y colorimétricas con sustratos específicos, y pruebas genotípicas (PCR convencional múltiple) para la detección simultánea de varios genes de resistencia (bla NDM, bla KPC, bla OXA-48-like, bla IMP y bla VIM). De los 128 aislamientos estudiados, 107 correspondieron a Klebsiella pneumoniae, 14 a Enterobacter cloacae complex, entre otros; aislados en mayor frecuencia de muestras de orina (30%), respiratorias (30%), sangre y catéter (24%). Los genes de resistencia a los carbapenemes detectados fueron: bla NDM (77,3%), bla KPC (17,2%); siendo confirmada la doble producción de carbapenemasas en 7 aislamientos (5,5%) provenientes de 4 centros diferentes de la capital de país y uno de Central; 6 de ellas (K. pneumoniae) con bla NDM+bla KPC y 1 (E. cloacae complex) con bla NDM+bla OXA-48-like; confirmando circulación de Enterobacterales dobles productores de carbapenemasas en el país (KPC+NDM y OXA+NDM); hallazgos que obligan a proveer de capacidades de detección, de manera a que se puedan tomar medidas oportunas y eficaces de contención y control.

ABSTRACT Carbapenemases are widely distributed in our country, both in fermenting and non-fermenting gram-negative bacilli. During 2021, an increase in strains with these enzymes has been reported. In order to evaluate the double production of carbapenemases in Enterobacterales and communicate their circulation, a multiple conventional PCR was set up. Retrospective study carried out in 128 isolates from 20 collaborating centers of the National AMR Surveillance Network (Capital, Central and interior of the country), sent to the LCSP between February and September 2021, for confirmation and genotyping of carbapenemases. Phenotypic and colorimetric tests were performed with specific substrates, as well as genotypic tests (multiple conventional PCR) for the simultaneous detection of several resistance genes (blaNDM, blaKPC, blaOXA-48-like, blaIMP and blaVIM). Of the 128 isolates studied, 107 corresponded to Klebsiella pneumoniae, 14 to Enterobacter cloacae complex, among others; isolated in higher frequency from urine (30%), respiratory (30%), blood and catheter (24%) samples. The genes for resistance to carbapenems detected were: blaNDM (77.3%), blaKPC (17.2%); the double production of carbapenemases was confirmed in 7 isolates (5.5%) from 4 different centers in the capital of the country and one in Central; 6 of them (K. pneumoniae) with blaNDM + blaKPC and 1 (E. cloacae complex) with blaNDM + blaOXA-48-like; confirming circulation of double Enterobacterales producers of carbapenemases in the country (KPC + NDM and OXA + NDM); findings that require the provision of detection capabilities, so that timely and effective containment and control measures can be taken.

Restricción programada de cefalosporinas de tercera generación en contexto de un brote de bacilos gramnegativos productores de beta-lactamasas tipo AmpC en unidades críticas: una experiencia de la vida real / Third-generation cephalosporins programmed restriction in the context of an outbreak of AmpC beta-lactamase-producing gram-negative bacilli in critical units: a real-life experience

INTRODUCCIÓN: La restricción programada (RP) de antimicrobianos puede disminuir selectivamente la tasa de infecciones por determinados microorganismos. En este sentido, los bacilos gramnegativos productores de beta-lactamasas AmpC (BGN-blaAmpC) son seleccionados por el sobreuso de cefalosporinas de tercera generación (C3G). Estas bacterias, también adquieren genes y co-producen otras beta-lactamasas, como las de Nueva Delhi (BGN-blaNDM). OBJETIVOS: Disminuir la tasa de aislamiento de BGN-blaAmpC y BGN-blaNDM en cultivos de pacientes de la UCI luego de una RP de C3G en el marco de un brote nosocomial por estos microrganismos. MATERIALES Y MÉTODOS: Estudio cuasi-experimental, previo (P1= 12 meses) y posterior (P2= 12 meses) a una RP de C3G en un hospital de adultos, donde, en el contexto de brote mencionado, se aplicaron medidas de control de infecciones generales. El uso de antimicrobianos se expresó como "porcentaje de los días de tratamiento (%DDT)"/100 camas ocupadas al día (100-COD). Se compararon las tasas de aislamiento de BGN-blaAmpC y BGN-blaNDM en hemocultivos (HC), mini-lavados bronquio-alveolares (mB) y urocultivos (UC) en la UCI. RESULTADOS: En P2 el consumo de C3G fue 2,5% DDT/100-COD. Hubo un descenso en los aislamientos de BGN-blaAmpC en HC (RR 0,48 [0,2-0,9] p < 0,02) y mB (RR 0,52 [0,3-0,9] p < 0,02), así como también de BGN-blaNDM en HC (RR 8,1 [1,6-39,4] p < 0,00). Conclusiones: La RP de C3G se asoció con la reducción de los BGN-blaAmpC y BGN-blaNDM en HC, así como de los BGN-blaAmpC mB.

BACKGROUND: Programmed restriction (PR) of antimicrobials can selectively decrease the rate of infections by certain microorganisms. In this sense, AmpC beta-lactamase-producing gram-negative bacilli (GNB-blaAmpC) are selected for the overuse of third generation cephalosporins (3GC). These bacteria also acquire genes and co-produce other β-lactamases, such as New Delhi ones (GNB-blaNDM). AIM: To decrease the isolation rate of GNB- blaAmpC and GNB- blaNDM in cultures from ICU patients after a PR of 3GC. METHODS: Quasi-experimental study, before (P1= 12 months) and after (P2= 12 months) a PR of 3GC in an adults' hospital. The use of antibiotics was expressed as "percentage days of treatment (%DOT)" /100 beds occupied per day (100-BOD). The rates of GNB-blaAmpC and GNB-blaNDM were compared in blood cultures (BC), mini-bronchio alveolar lavages (mB) and urine cultures (UC) in the ICU. RESULTS: In P2, 3GC consumption was 2.5% DOT/100-COD. There was a decrease in GNB-blaAmpC from BC (RR 0.48 [0.2-0.9] p < 0.02) and mB (RR 0.52 [0.3-0.9] p < 0.02), as well as of GNB-blaNDM from BC (RR 8.1 [1.6-39.4] p < 0.00). Conclusions: PR of 3GC was linked to the reduction of GNB-blaAmpC and GNB-blaNDM in BC, as well as GNB-blaAmpC in mB from ICU patients.

Detección fenotípica y genotípica de la producción de carbapenemasas tipo NDM-1 y KPC en enterobacterias aisladas en un laboratorio clínico en Maracay, Venezuela / Phenotypic and genotypic detection of the production of carbapenemases type NDM-1 and KPC in isolated Enterobacteriaceae in a clinical laboratory in Maracay, Venezuela

INTRODUCCIÓN: La producción de beta-lactamasas capaces de hidrolizar a los carbapenémicos es uno de los mecanismos de resistencia más preocupantes porque eliminan la última opción terapéutica frente a los microorganismos multi-resistentes. OBJETIVO: Determinar la producción de carbapenemasas tipo KPC y NDM-1, empleando métodos fenotípicos y genotípicos, en enterobacterias aisladas en un laboratorio clínico de la ciudad de Maracay, Venezuela. MÉTODOS: Se determinó la producción de carbapenemasas mediante métodos fenotípicos (según algoritmo de Malbrán) y genotípicos (amplificación de los genes blaNDM-1 y blaKPC por RPC) en enterobacterias aisladas en un laboratorio clínico durante el período marzo-agosto 2018. RESULTADOS: Se identificaron 605 enterobacterias de diferentes especies, siendo Escherichia coli la cepa con mayor porcentaje de aislamiento (61,3%), seguida por Klebsiella pneumoniae (14,9%). Diez y seis enterobacterias (2,64%) fueron positivas para la producción de carbapenemasas: 13 cepas de K. pneumoniae y tres del complejo Enterobacter cloacae. La RPC demostró que 14 cepas (87,5%) contienen el gen blaNDM-1 y dos (12,5%) el gen blaKPC; se observó 100% de concordancia entre la determinación fenotípica y la RPC para ambos grupos de enzimas. CONCLUSIONES: Los resultados mostraron mayor incidencia de la metalo-beta-lactamasa tipo NDM-1, reconocida como una alarma epidemiológica debido a que su rápida diseminación dificulta su control, por lo que la identificación del tipo de enzima permitiría establecer estrategias de manejo y control más certeras con la finalidad de erradicar a dichos patógenos.

BACKGROUND: The production of carbapenem-hydrolyzing beta-lactamases is one of the most concerning resistance mechanisms since it eliminates the last therapeutic option against multidrug resistant microorganisms. AIM: To determine the production of KPC and NDM-1 type carbapenemases, using phenotypic and genotypic methods, in isolated enterobacteria in a clinical laboratory in the city of Maracay, Venezuela. METHODS: The production of carbapenemases was determined by phenotypic (according to the Malbrán algorithm) and genotypic methods (amplification of the blaNDM-1 and blaKPC genes by PCR) in clinical isolates of Enterobacteriaceae during the period March-August 2018. RESULTS: 605 Enterobacteriaceae of different species were identified, being Escherichia coli the strain with the highest percentage of isolation (61.3%), followed by Klebsiella pneumoniae (14.9%). Sixteen strains (2.64%) were positive for carbapenemases production: 13 strains of K. pneumoniae and three of the Enterobacter cloacae complex. PCR showed that 14 strains (87.5%) carry the blaNDM-1 gene and two strains (12.5%) the blaKPC gene; 100% agreement was observed between phenotypic determination and PCR for both groups of enzymes. CONCLUSIONS: The results of this study showed a higher incidence of metallo-beta-lactamase type NDM-1, which rapid dissemination and consequently difficult control has been cause of epidemiological alert. The identification of the type of enzyme would allow establishing more accurate management and control strategies in order to eradicate these pathogens.

First report of a blaNDM-resistant gene in a Klebsiella aerogenes clinical isolate from Brazil

Abstract INTRODUCTION: Carbapenemase-resistant enterobacteria that produce the bla NDM gene are found worldwide. However, this is the first report of blaNDM in Klebsiella aerogenes in Brazil. METHODS: The identification of bacterial species was performed using anautomated system and confirmed by biochemical tests, 16S rRNA gene sequencing, and detection of resistance genes. RESULTS: The clinical isolate showed minimum inhibitory concentration resistance to meropenem and polymyxin B at 8mg/L and 4mg/L, respectively. Only the blaNDM gene was detected. CONCLUSIONS: The current report of the blaNDM gene in isolated MDR enterobacteria indicates that this gene can spread silently in a hospital setting.

First report of bla GES-1 in Proteus mirabilis clinical isolates

Abstract Proteus mirabilis is one of the main pathogens causing urinary tract infections and sepsis. To our knowledge, this is the first report of a P. mirabilis hosting bla GES. The presence of these genes was determined using PCR and sequencing. We identified the presence of bla GES-1 in all three isolates. In addition, we identified the bla KPC-2 and bla NDM-1 genes in the two strains. These data emphasize the importance of monitoring and surveillance of all enterobacteria. The circulation of P. mirabilis strains carrying bla GES-1 constitutes a new scenario of resistance in this species and should be an epidemiological alert for global health.

Páramos Neotropicales como unidades biogeográficas

Introducción: El Páramo se ha definido desde diversos acercamientos, teniendo en cuenta factores de fácil reconocimiento o medición. A nivel biogeográfico se ha evaluado con métodos ampliamente criticados para la identificación de áreas de endemismo. El análisis de endemicidad, pese a su importancia y amplio reconocimiento, no se ha utilizado como herramienta para evaluar el Páramo. Objetivo: Determinar si los páramos neotropicales es una o varias unidades biogeográficas. Métodos: Incluimos registros de Aves, Amphibia, Mammalia, Reptilia, Marchantiophyta y Spermatophyta, para los que encontramos 7 025 especies con 193 250 presencias viables obtenidas desde GBIF (Global Biodiversity Information Facility) [a septiembre de 2018]. Usamos cada grupo taxonómico como una partición independiente, y generamos particiones adicionales como plantas totales (Plantas-T: Marchantiophyta + Spermatophyta), animales totales (Animales-T: Aves + Amphibia + Mammalia + Reptilia) y evidencia total (Plantas-T + Animales-T). Utilizamos el criterio de optimalidad para identificar áreas de endemismo. Realizamos el análisis usando dos tamaños de cuadrícula 0.5 y 0.25°. Con las áreas obtenidas, calculamos la intersección con los polígonos que representan las definiciones de páramo generadas por otros autores. Resultados: Con los dos tamaños de cuadrícula identificamos áreas de endemismo en diferentes sectores; sin embargo, el tamaño de 0.25° nos permitió mayor resolución al identificar los sectores en alta montaña. Estos sectores corresponden a ocho zonas que denominamos subprovincias: Santa Marta-Perijá, Mérida, Santanderes-Boyacá, Cundinamarca, Cordillera Central-Occidental, Norte de Ecuador, Centro-Sur de Ecuador y Talamanca, las cuales fueron congruentes entre un 4 y un 66 % con las definiciones previas. Conclusiones: Páramo se ha planteado como una sola unidad biogeográfica; sin embargo, dado nuestros análisis, lo identificamos como ocho subprovincias biogeográficas, congruentes con estudios previamente publicados.

Neotropical páramos as biogeographic units. Introduction: Páramo has been defined from various points of view, which take into account different factors that are easy to recognize or measure, nevertheless at the biogeographic level it has been evaluated with criticized methods used to identify historical units. The analysis of endemicity, despite its importance and wide recognition, has not been used as a tool to evaluate Páramo. Objective: Determine whether the neotropical Páramo is one or several biogeographic units. Methods: We included distributional records from Aves, Amphibia, Mammalia, Reptilia, Marchantiophyta, and Spermatophyta. We found 7 025 species with 193 250 suitable occurrences obtained from the GBIF. We used each taxonomic group as an independent partition or as a component of a larger partition, such as total plants (Plants-T: Marchantiophyta + Spermatophyta), or total animals (Animals-T: Aves + Amphibia + Mammalia + Reptilia), or total evidence (Plants-T + Animals-T). In order to identify areas of endemism, we used the optimality criterion (NDM/VNDM) with grids of 0.5° or 0.25°. We calculated the intersection among polygons of previous definitions and the areas recovered in our analyses. Results: Both grid sizes, 0.25° and 0.5°, identified areas of endemism in different sectors along the Andean and Central American cordilleras, but only the 0.25° size allowed us to recognize areas/sectors with a higher resolution. We recovered eight areas, which were considered as subprovinces (Santa Marta-Perijá, Mérida, Santanderes-Boyacá, Cundinamarca, Central-Western Cordillera, Northern Ecuador, Central-South Ecuador, and Talamanca). These areas were between 4 and 66 % consistent with previous definitions. Conclusions: Páramo has been considered a single biogeographic unit, however, given our analyses we identified it as a unit composed of eight biogeographic subprovinces, which is consistent with some published studies.

Evaluation of Xpert Carba-R Assay v.2 to Detect Carbapenemase Genes in Two Hospitals in Korea

BACKGROUND: As the spread of carbapenemase-producing Enterobacteriaceae poses a critical threat to public health, rapid detection of carbapenemase genes is urgently required for prompt initiation of appropriate antimicrobial therapy and infection control. We evaluated the performance of Xpert Carba-R v.2 (Cepheid, USA) compared with that of culture-based conventional PCR.METHODS: Using the results of 5,479 consecutive clinical rectal swabs, discrepant analysis (enriched culture followed by PCR) was performed for all discordant samples (N=100), which were Carba-R v.2-positive and culture-negative.RESULTS: Among the samples, 206 carbapenemase genes (3.6%) were detected by Carba-R v.2. The sensitivity and specificity were 95.0% and 98.1%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 49.0% and 99.9%, respectively. Following discrepant analysis, the PPV increased to 73.5% and the low PPV (8.1%) of the 86 non-KPC improved to 48.8%. Among the 105 discrepancies, NDM was the most frequently observed (N=56), followed by KPC (N=26), VIM (N=10), IMP (N=8), OXA-48 (N=5). The threshold cycle values between discordant vs. concordant and resolved groups were significantly different (P<0.001).CONCLUSIONS: Carba-R v.2 is a rapid and sensitive method for detecting carbapenemase-encoding genes compared with culture-based conventional PCR. Most of our discrepant results were non-KPC genes. Thus, the clinical significance of the non-KPC positive cases detected by Carba-R v.2 should be investigated. This assay would be useful for deciding whether to isolate pre-exposed patients in hospital settings, based on the high specificity and NPV.

Rapid Identification of OXA-48-like, KPC, NDM, and VIM Carbapenemase-Producing Enterobacteriaceae From Culture: Evaluation of the RESIST-4 O.K.N.V. Multiplex Lateral Flow Assay

There is an urgent need for accurate and rapid diagnostic assays capable of identifying carbapenemase-producing Enterobacteriaceae (CPE). We assessed the performance of the RESIST-4 O.K.N.V. (OKNV) assay (Coris BioConcept, Gembloux, Belgium) for the identification of oxacillinase (OXA)-48-like-, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-β-lactamase (NDM)-, and Verona integron-encoded metallo-β-lactamase (VIM)-producing Enterobacteriaceae grown on sheep blood agar (SBA) and the CHROMagar KPC medium. Sixty-five carbapenem-resistant Enterobacteriaceae (CRE) isolates with characterized carbapenemase content were used to evaluate the OKNV assay. The assay correctly identified all 30 isolates that produced one of the four targeted carbapenemase families. Additionally, it correctly identified 15 isolates that co-produced KPC and NDM, VIM and NDM or OXA-48-like and NDM, but failed to identify an NDM-1 and OXA-232 co-producing Klebsiella pneumoniae isolate. All 16 non-carbapenemase-producing CRE and four CPE isolates exhibited negative results, and no cross-reaction was observed. Overall, the sensitivity and specificity of the assay were 97.8% and 100%, respectively. The OKNV assay is an accurate and rapid assay for identifying OXA-48-like, KPC, NDM, and VIM carbapenemases produced by Enterobacteriaceae isolates cultured on both SBA and the CHROMagar KPC media in the clinical microbiology laboratory.

Will ceftazidime/avibactam plus aztreonam be effective for NDM and OXA-48-Like producing organisms: Lessons learnt from In vitro study

Introduction: Carbapenem resistance (CR) in Klebsiella pneumoniae is mainly mediated by bla NDM and bla OXA-48 carbapenemases. Newer Food and Drug Administration-approved antimicrobial ceftazidime/avibactam (C/A) has a potent activity against bla OXA-48-like producers. However, its activity is limited in organisms co-producing bla NDM and bla OXA-48-like. Addition of aztreonam (ATM) to C/A potentially expands the spectrum of coverage for carbapenemase co-producers. With this, we aimed to determine the synergistic activity of combination of C/A plus ATM against bla NDM, bla OXA-48-like and co-producers of bla NDM + bla OXA-48-like producing CR Klebsiella pneumoniae (CRKp). Materials and Methods: A total of 12 isolates of CRKp-harbouring genes encoding bla NDM and bla OXA-48-like were tested. Minimum inhibitory concentrations (MICs) were determined for several antimicrobial agents, including C/A (0.5�?g/ml) by broth microdilution method. Checkerboard assay was performed for the combination of C/A plus ATM at varying concentrations. Fold differences in the MIC of C/A with and without addition of ATM were determined to infer synergistic effects. Results: MIC of C/A and ATM ranged from 0.5 to >8 ?g/ml and 64 to 2048 ?g/ml, respectively. Two isolates were susceptible to C/A with MIC of 0.5 and 1 ?g/ml, while others were resistant with MIC of >8 ?g/ml. Synergistic effects of >8-fold MIC difference in C/A MIC were noted with addition of ATM at 4 ?g/ml. This was observed for all CRKp with profiles of bla NDM, bla OXA-48-like and co-producers of bla NDM + bla OXA-48-like genes, which was a promising effect. Notably, all five of the colistin-resistant CRKp were inhibited with >8-fold MIC difference in the combination of C/A plus ATM at 4 ?g/ml. Conclusion: With the increasing burden of CRKp, the use of C/A with ATM combination seems to be very promising, especially for bla NDM, bla OXA-48-like and co-producers of bla NDM + bla OXA-48like carbapenemases.

Colistin-sparing approaches with newer antimicrobials to treat carbapenem-resistant organisms: Current evidence and future prospects

Antimicrobial resistance is on the rise across the globe. Increasing incidence of infections due to carbapenem resistance organisms is becoming difficult to treat, due to the limited availability of therapeutic agents. Very few agents such as colistin, fosfomycin, tigecycline and minocycline are widely used, despite its toxicity. However, with the availability of novel antimicrobials, beta-lactam/beta-lactamase inhibitor-based and non-beta-lactam-based agents could be of great relief. This review covers three important aspects which include (i) current management of carbapenem-resistant infections, (ii) determination of specific types of carbapenemases produced by multidrug-resistant and extensively drug-resistant Gram-negative pathogens and (iii) the currently available novel beta-lactam/beta-lactamase inhibitors and non-beta-lactam-based agents' laboratory findings, clinical outcome and implications.

Blandm-1 Gene Carriage In The Icu Of Tertiary Care Hospital In North India

BACKGROUND:- New Delhi metallo-β-lactamase 1 (NDM-1) enzyme leads to multidrug resistance and has been detected from bacteria in many countries. The study was done To detect bacteria carrying blaNDM-1 gene from intensive care unit (ICU) patients and correlate the change with increasing duration of ICU stay. METHODS:- Blood and urine samples were collected from 140 patients thrice- 0-2 days, 3-7 days and 7 days of ICU admission. All bacterial isolates resistant to meropenem were evaluated for Metallo-β-lactamase (MBL) production. blaNDM-1 gene was detected using Real Time PCR from the MBLproducers. RESULT:- Overall, 458 samples (229 each) of blood and urine were collected and 75 gram negative bacteria were isolated. From these, 46.7% (35/75) strains were found to be carbapenem resistant and blaNDM-1 gene was detected in 17.3% (13/75). CONCLUSION:- High prevalence of bacteria carrying blaNDM-1 gene was seen in ICU patients increasing the burden on healthcare.

Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria

Background & objectives: The growing incidence and the wide diversity of carbapenemase-producing bacterial strains is a major concern as only a few antimicrobial agents are active on carbapenem-resistant bacteria. This study was designed to study molecular epidemiology of carbapenem-resistant Gram-negative bacterial (GNB) isolates from the community and hospital settings. Methods: In this study, non-duplicate GNB were isolated from clinical specimens, and phenotypic test such as modified Hodge test, metallo ?-lactamase E-strip test, etc. were performed on carbapenem-resistant bacteria. Multiplex PCR was performed to identify the presence of blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM. Minimum inhibitory concentration (MIC) of colistin, fosfomycin, minocycline, chloramphenicol and tigecycline was also determined. Results: Of the 3414 GNB studied, carbapenem resistance was 9.20 per cent and maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4%) and primary centre (2.1%). Among the carbapenem-resistant bacteria, overall, the most common isolate was Pseudomonas aeruginosa (24%). On multiplex PCR 90.3 per cent carbapenem-resistant isolates were positive for carbapenemase gene. The blaNDM(63%) was the most prevalent gene followed by blaVIM(18.4%). MIC results showed that 88 per cent carbapenem-resistant Enterobacteriaceae were sensitive to fosfomycin, whereas 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin. Interpretation & conclusions: Carbapenem resistance in GNB isolates from the community and hospital settings was found to be on the rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, prudent use of antibiotics and strict infection control practices should be followed in hospital to limit the emergence and spread of multidrug-resistant bacteria.