Antimicrobial resistance and associated risk factors in Escherichia coli isolated from Peruvian dogs: A focus on extended-spectrum ß-lactamases and colistin.

Background and Aim: Established antimicrobial resistance (AMR) surveillance in companion animals is lacking, particularly in low-middle-income countries. The aim of this study was to analyze AMR and its risk factors in Escherichia coli isolated from dogs at two veterinary centers in Lima (Peru). Materials and Methods: Ninety dogs were included in the study. Antimicrobial susceptibility was established by disk diffusion, whereas microdilution was used to determine colistin susceptibility. Mechanisms related to extended-spectrum ß-lactamases (ESBL) and colistin resistance were determined by polymerase chain reaction. Clonal relationships of colistin-resistant isolates were assessed by XbaI-pulsed-field gel electrophoresis. Results: Thirty-five E. coli strains were isolated. High levels of resistance to ampicillin (57.1%), nalidixic acid (54.3%), tetracycline (48.6%), and azithromycin (25.7%) were detected. Cephalosporin resistance levels were ≥20% and those for colistin were 14.3%. Twelve (34.2%) isolates were ESBL producers; of these, six blaCTX-M-55 (50.0%), 2 (16.6%) blaCTX-M-15, and 2 (16.6%) blaCTX-M-8-like genes were found. The five colistin-resistant isolates were clonally unrelated, with four of them presenting amino acid codon substitutions in the mgrB gene (V8A) or mutations in the mgrB promoter (a12g, g98t, and c89t). Furthermore, dog age, <6 years (p = 0.027) and raw diet (p = 0.054) were associated with resistance to a greater number of antibiotic families. Conclusion: Despite small number of samples included, the study found that dogs studied were carriers of multidrug-resistant E. coli, including last-resort antimicrobials, representing a public health problem due to close contact between dogs and humans. This issue suggests the need for larger studies addressed to design strategies to prevent the spread of resistant micro-organisms in small animal clinics and domestic settings.

Prevalence and characteristics of ESBL-producing Escherichia coli in clinically healthy pigs: implications for antibiotic resistance spread in livestock.

AIM: This study aimed to compare and characterize the resistance profile and the presence of extended-spectrum beta-lactamase (ESBL) related genes in Escherichia coli isolated from healthy finishing pigs fed with or without antibiotics in their diets. METHODS AND RESULTS: A total of 27 ceftiofur-resistant E. coli isolates were obtained from 96 healthy pigs. The antibiotic resistance profile was tested, and all 27 isolates were classified as multidrug-resistant (MDR). A high proportion of isolates were resistant to cephalosporins, ampicillin, ciprofloxacin, and tetracyclines. The ESBL production was observed in 85% of isolates by double-disc synergy test. The MDR-E. coli isolates harbored ESBL genes, such as blaTEM, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-8,25. In addition, other antibiotics resistance genes (ARGs) were also detected, such as sul2, ant(3″)-I, tetA, and mcr-1. The mobilization of the blaCTX-M gene was confirmed for nine E. coli isolates by conjugation assays. The presence of blaCTX-M on mobile genetic elements in these isolates was demonstrated by Southern blot hybridization, and the resistance to cephalosporins was confirmed in the transconjugants. Our results indicate the prevalence of CTX-M-producing E. coli strains harboring mobile genetic elements in the normal microbiota of healthy pigs. CONCLUSIONS: These findings highlight the significance of ESBL genes as a global health concern in livestock and the potential spread of antimicrobial resistance to other members of the gastrointestinal tract microbiota.

Clinical outcomes of intensive care unit patients infected with multidrug-resistant gram-negative bacteria treated with ceftazidime/avibactam and ceftolozane/tazobactam.

In intensive care units (ICUs), infection rates range from 18 to 54%, which is five to ten times higher than those observed in other hospital units, with a mortality rate of 9% to 60%. In recent decades, the susceptibility pattern has changed and Gram-Negative Bacteria (GNB) have become a threat due to their high frequency of multidrug resistance associated with a scarcity of therapeutic options. However, the drugs Ceftolozane/Tazobactam (C/T) and Ceftazidime/Avibactam (C/A) are demonstrating good clinical and microbiological response in the treatment of severe nosocomial infections. Therefore, this study aims to evaluate the clinical outcome of patients with severe infections caused by Multidrug-Resistant (MDR) GNB treated with C/T and C/A. Our study evaluates a total of 131 patients who received treatment with C/T and C/A due to infections caused by MDR GNB within the period from 2018 to 2021. The main infections were urinary tract (46,6%) and respiratory (26,7%) infections. Pseudomonas aeruginosa was the prevailing agent in the sample evaluation (34.3%), followed by Klebsiella pneumoniae (30,1%). About 54,9% of patients showed a favorable response, with culture negativation in 66,4% of the samples, with no discrepancy in negativations when comparing ages: 67,7% in young and 66% in elderly patients. Among the patients, 62,6% received monotherapy with C/T and C/A with a better response observed with monotherapy compared to combination therapy (58,6% vs 41,4%). The overall mortality rate was 45%, with MDR GNB infections responsible for 33,9% of these deaths, and the others (66,1%) due to factors such as oncological, hematological, and degenerative neurological diseases. In regards to hematological aspect, 35,1% of patients showed changes, with 28,2% of them presenting anemia, 4,5% thrombocytopenia, and 2,5% thrombocytosis. Concerning the use of invasive devices, higher mortality was observed in patients on mechanical ventilation (52%). In this manner, it was possible to observe that therapy with C/T and C/A yielded a favorable clinical outcome in patients with severe infections caused by MDR GNB in the study. These drugs also demonstrated good tolerability regardless of age or the presence of preexisting comorbidities and were deemed safe when assessing adverse effects. Our data also demonstrate the importance of determining the mechanism of resistance to carbapenems so that these drugs can be used more effectively and rationally.

Molecular mechanism of antimicrobial co-resistance Colistin (mcr-1) and ESBLs genes among Escherichia coli isolates from commercial chickens in Pakistan / Mecanismo molecular de corresistência antimicrobiana de Colistina (mcr-1) e genes ESBLs entre isolados de Escherichia coli em frangos comerciais no Paquistão

Emergence of plasmid mediated colistin and extended spectrum ß-lactamases (ESBL) resistant genes has been impacted the efficacy of colistin and ß-lactams drugs like 3rd, 4th generation cephalosporin. Current study was aimed to investigate antimicrobial resistance genes (ARGs) among Escherichia coli isolates from meat producing commercial broilers in Pakistan. Two hundred (n=200) fecal samples were collected during January-2018 to August-2019. For isolation of E. coli, pink colonies on MacConkey agar were transferred to EMB agar. Metallic sheen color colonies were tested biochemically using API-20E kit. The molecular identification of E. coli (n=153) was targeted by amplification of uid gene through polymerase chain reaction (PCR) and different ARGs i.e. gentamicin, streptomycin, tetracycline, colistin, ß-lactams drugs, quinolone and ampicillin followed by sequence analysis. Genotypically, followed by phenotypically of resistant ARGs of isolated PCR-confirmed E. coli (153) shoed resistant against gentamicin (aac(3)-IV), streptomycin (aadA1), tetracycline (tetA), colistine (mcr-1), ampicillin (bla-TEM) and bla-CTX-M were 86%, 88%, 86%, 88%, 83% & 77% respectively. 33/38 (86%) of the isolate was positive for quinolone resistance. Colistine (mcr-1), ESBLs (bla-TEM) and (bla-CTX-M) resistance genes were 88%, 83% and 77% respectively. About 33 isolated E. coli harbored the both mcr-1 and ESBLs genes. All of E. coli isolates were found sensitive to ceftriaxone (CTX-30) and imipenem (IMP-10). The Isolated E. coli showed single or multi clade decadency. The E. coli and ARGs sequences showed single or multi clade decadency. This is first comprehensive study from Pakistan that described the molecular evidences of ARGs and their co-existence in single isolates originated from commercial poultry. Commercial chicken (Broilers) can act as melting pot of antibiotic resistance genes for human being. It is alarming situation for surveillance of antibiotic resistance program because of more regulated prescription of antimicrobial agents in Pakistan.

O surgimento de colistina mediada por plasmídeo e genes de resistência a ß-lactamases de espectro estendido (ESBL) afetou a eficácia de medicamentos colistina e ß-lactâmicos, como as cefalosporinas de 3ª e 4ª geração. O presente estudo teve como objetivo investigar genes de resistência antimicrobiana (ARGs) entre isolados de Escherichia coli em frangos de corte comerciais no Paquistão. Duzentas (n = 200) amostras fecais foram coletadas durante janeiro de 2018 a agosto de 2019. Para o isolamento de E. coli, colônias rosas em ágar MacConkey foram transferidas para ágar EMB. As colônias de cores de brilho metálico foram testadas bioquimicamente usando o kit API-20E. A identificação molecular de E. coli (n = 153) foi direcionada pela amplificação do gene uid através da reação em cadeia da polimerase (PCR) e diferentes ARGs, ou seja, gentamicina, estreptomicina, tetraciclina, colistina, medicamentos ß-lactâmicos, quinolona e ampicilina, seguido de análise de sequência. Genotipicamente, seguido por fenotipicamente de ARGs resistentes de E. coli isoladas foram confirmadas por PCR (153) como resistente contra gentamicina (aac(3)-IV), estreptomicina (aadA1), tetraciclina (tetA), colistina (mcr-1), ampicilina (bla-TEM) e bla-CTX-M, demonstrando resultados de 86%, 88%, 86%, 88%, 83% e 77%, respectivamente. Cerca de 33/38 (86%) do isolado foi positivo para resistência às quinolonas. Os genes de resistência à colistina (mcr-1), ESBLs (bla-TEM) e (bla-CTX-M) foram 88%, 83% e 77%, respectivamente. Cerca de 33 E. coli isoladas continham os genes mcr-1 e ESBLs. Todos os isolados de E. coli foram considerados sensíveis à ceftriaxona (CTX-30) e imipenem (IMP-10). A E. coli isolada apresentou decadência de um ou vários clados. As sequências de E. coli e ARGs apresentaram decadência de um ou vários clados. Este é o primeiro estudo abrangente do Paquistão que descreveu as evidências moleculares de ARGs e sua coexistência em isolados únicos originados de aves comerciais. Dessa forma, é possível concluir que o Frango comercial (Broilers) pode atuar como caldeirão de genes de resistência a antibióticos para o ser humano. É uma situação alarmante para a vigilância do programa de resistência a antibióticos devido à prescrição mais regulamentada de agentes antimicrobianos no Paquistão.

Traditional marketed meats as a reservoir of multidrug-resistant Escherichia coli.

This study aimed to analyze Escherichia coli from marketed meat samples in Peru. Sixty-six E. coli isolates were recovered from 21 meat samples (14 chicken, 7 beef), and antimicrobial resistance levels and the presence of mechanisms of antibiotic resistance, as well as clonal relationships and phylogeny of colistin-resistant isolates, were established. High levels of antimicrobial resistance were detected, with 93.9% of isolates being multi-drug resistant (MDR) and 76.2% of samples possessing colistin-resistant E. coli; of these, 6 samples from 6 chicken samples presenting mcr-1-producer E. coli. Colistin-resistant isolates were classified into 22 clonal groups, while phylogroup A (15 isolates) was the most common. Extended-spectrum ß-lactamase- and pAmpC-producing E. coli were found in 18 and 8 samples respectively, with blaCTX-M-55 (28 isolates; 16 samples) and blaCIT (8 isolates; 7 samples) being the most common of each type. Additionally, blaCTX-M-15, blaCTX-M-65, blaSHV-27, blaOXA-5/10-like, blaDHA, blaEBC and narrow-spectrum blaTEM were detected. In addition, 5 blaCTX-M remained unidentified, and no sought ESBL-encoding gene was detected in other 6 ESBL-producer isolates. The tetA, tetE and tetX genes were found in tigecycline-resistant isolates. This study highlights the presence of MDR E. coli in Peruvian food-chain. The high relevance of CTX-M-55, the dissemination through the food-chain of pAmpC, as well as the high frequency of unrelated colistin-resistant isolates is reported.

Characterization of ESBL-producing Escherichia spp. and report of an mcr-1 colistin-resistance Escherichia fergusonni strain from minced meat in Pamplona, Colombia.

Foods of animal origin are increasingly considered a source of extended spectrum ß-lactamase (ESBL) producing bacteria which can disseminate throughout the food chain and become a health concern for humans. This work aimed to evaluate the occurrence of ESBL-producing Escherichia coli in 100 retail minced meat samples taken in markets in Pamplona, Colombia. A total of 19 ESBL-producing isolates were obtained, 18 identified as E. coli and one as E. fergusonii. Fifteen isolates (78.9 %) carried blaCTX-M and blaTEM genes, one (5.2 %) blaSHV and blaTEM genes, one isolate (5.2 %) carried blaCTX-M and one (5.2 %) blaSHV alone. The majority of CTX-M-positive E. coli isolates carried the blaCTX-M-15 gene (13 isolates), being the blaCTX-M-9, blaCTX-M-2, and blaCTX-M-8 (one isolate each) also detected. Two SHV-positive isolates presented the blaSHV-5 and blaSHV-12 allele. The isolate identified as E. fergusonii was positive for blaCTX-M-65 gene and mcr-1 gene. Sixteen isolates (84.2 %) belonged to phylogroups A and B1 and grouped together in the phylogenetic tree obtained by MLST; phylogroups E and F were also detected. Transfer of ESBL resistance was demonstrated for the E. fergusonii isolate. Whole genome sequencing of this isolate revealed the presence of plasmids carrying additional resistance genes. This investigation showed the high prevalence of ESBL-producing E. coli in retail samples of minced meat. Also, the isolation of a strain of E. fergusonii is an additional concern, as some resistance genes are located in mobile elements, which can be transmitted to other bacteria. These evidences support the increasing public health concern considering the spreading of resistance genes through the food chain.

High Prevalence of blaCTX-M in Fecal Commensal Escherichia coli from Healthy Children.

BACKGROUND: Antibiotic-resistant Escherichia coli can colonize the intestinal tract of healthy children, causing concern when antibiotic resistance is related to the presence of transferable mechanisms, such as extended-spectrum ß-lactamases (ESBLs). MATERIALS AND METHODS: Fecal samples from 41 healthy children from two villages of rural Peru were cultured on ceftriaxone-disks. ESBL production was confirmed with double disk synergy. In all ESBL-produced isolates, antibiotic susceptibility to 12 antibacterial agents was established by disk diffusion, while clonal relationships were determined by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). Presence of ST131 was determined using PCR. RESULTS: Ceftriaxone-resistant microorganisms were recovered from 39 samples belonging to 22 out of 41 children (53.7%). Of these, 80 ceftriaxone-resistant and two ceftriaxone-intermediate E. coli from inside ceftriaxone-halos were confirmed as ESBL-producers. All isolates were multidrug-resistant. In 79/80 (98.8%) ceftriaxone-resistant isolates, the presence of blaCTX-M was detected alone (58 isolates, or together with other ß-lactamase (blaTEM, 17 isolates; blaOXA-1-like, 3 isolates; blaTEM + blaOXA-1-like, 1 isolate), while in one isolate no such ESBL was identified. The two ceftriaxone-intermediate isolates recovered from the same sample, carried a blaTEM and blaSHV respectively. Thirty-four different clones were identified, with 4 clones being recovered from different samples from the same child. Twelve clones were disseminated among different children, including 5 clones disseminated between both villages. Two clones, accounting for 3 isolates and both recovered from the same children, belonged to E. coli ST131. CONCLUSION: This study demonstrates high prevalence of ESBL-carriers among healthy children living in a rural area of Peru, stressing the need for continuous surveillance and search for public health control measures.

Colistin Heteroresistance among Extended Spectrum ß-lactamases-Producing Klebsiella pneumoniae.

Colistin-heteroresistant (CST-HR) Enterobacterales isolates have been identified recently, challenging the clinical laboratories since routine susceptibility tests fail to detect this phenotype. In this work we describe the first CST-HR phenotype in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolates in South America. Additionally, we determine the genomic mechanisms of colistin heteroresistance in these strains. The CST-HR phenotype was analyzed by the population analysis profile (PAP) method, and mutations associated with this phenotype were determined by whole-genome sequencing (WGS) and the local BLAST+ DB tool. As a result, 8/60 isolates were classified as CST-HR according to the PAP method. From WGS, we determined that the CST-HR isolates belong to three different Sequence Types (STs) and four K-loci: ST11 (KL15 and KL81), ST25 (KL2), and ST1161 (KL19). We identified diverse mutations in the two-component regulatory systems PmrAB and PhoPQ, as well as a disruption of the mgrB global regulator mediated by IS1-like and IS-5-like elements, which could confer resistance to CST in CST-HR and ESBL-producing isolates. These are the first descriptions in Chile of CST-HR in ESBL-producing K. pneumoniae isolates. The emergence of these isolates could have a major impact on the effectiveness of colistin as a "last resort" against these isolates, thus jeopardizing current antibiotic alternatives; therefore, it is important to consider the epidemiology of the CST-HR phenotype.

The crystal structure of ESBL TLA-1 in complex with clavulanic acid reveals a second acylation site.

ß-lactamases are the main molecules responsible for giving bacterial resistance against ß-lactam antibiotics. The study of ß-lactamases has allowed the development of antibiotics capable of inhibiting these enzymes. In this context, extended spectrum ß-lactamase (ESBL) TLA-1 has spread in Escherichia coli and Enterobacter cloacae clinical isolates during the last 30 years in Mexico. In this research, the 3D structures of ESBL TLA-1 and TLA-1 S70G mutant, both ligand-free and in complex with clavulanic acid were determined by X-ray crystallography. Four clavulanic acid molecules were found in the structure of TLA-1, two of those were intermediaries of the acylation process and were localized covalently bound to two different amino acid residues, Ser70 and Ser237. The coordinates of TLA-1 in complex with clavulanic acid shows the existence of a second acylation site, additional to Ser70, which might be extendable to several members of the subclass A ß-lactamases family. This is the first time that two serines involved in binding clavulanic acid has been reported and described to an atomic level.

Detección de los genes de ß-lactamasas blaTEM, blaSHV y blaCTX-M en aislamientos de Escherichia coli comunitarios / Detection of ß-lactamase genes bla TEM, bla SHV and bla CTX-M in community isolates of Escherichia coli

A nivel mundial la resistencia a los antibióticos es un problema de salud pública, tanto en el ámbito hospitalario como en el comunitario. La producción de ß-lactamasas es el principal mecanismo de resistencia en enterobacterias y la mayoría de enzimas responsables pertenecen a las familias TEM, SHV y CTX-M. El objetivo de este estudio fue detectar los genes de ß-lactamasas blaTEM, blaSHV y blaCTX-M en cepas comunitarias de Escherichia coli productoras de BLEE aisladas de urocultivos de pacientes que acudieron al Laboratorio Clínico Popular de la Universidad de San Carlos de Guatemala en el año 2016. Se detectó la presencia de al menos uno de los genes en el 90% de los 79 aislamientos y un 53.2% presentó los tres genes. La frecuencia fue de 57% para blaCTX-M, 84% para bla SHV y 85% para blaTEM. La detección de los genes codificadores de las enzimas TEM-1, SHV-11, CTX-M15 y CTX-M55 corresponde a la primera caracterización molecular de aislamientos de E. coli productoras de BLEE en Guatemala y son importantes para entender su propagación en el ámbito comunitario. Los aislamientos de E. coli productoras de BLEE mostraron alta resistencia a ciprofloxacina y trimetoprim sulfametoxazol (78%) y bajos niveles de resistencia para fosfomicina (2.5%) y nitrofurantoina (7.6%). El 11.39% de las cepas presentó resistencia a un grupo de antibióticos no betalactámicos. Es importante establecer una vigilancia activa para la resistencia de estos antibióticos en cepas comunitarias ya que son la primera opción de tratamiento para cepas productoras de BLEE

Globally, resistance to antibiotics is a public health problem, both in the hospital and in the community environment. e production of ß-lactamases is the main mechanism of resistance in enterobacteria and usually the cause of resistance are the enzymes to the families TEM, SHV and CTX-M. e principal aim of this study was to detect - ß-lactamase genes blaTEM, blaSHV and blaCTX-M in community strains of ESBL-producing Escherichia coli isolated from urine cultures of patients attended in the Laboratorio Clínico Popular of the Universidad de San Carlos de Guatemala in 2016. At least, one of the genes was detected in 90% of the 79 isolates and in 53.2% of the isolates the three genes were detected. e frequency was 57% for blaCTX-M, 84% for blaSHV and 85% for blaTEM. e detection of the genes coding for TEM-1, SHV-11, CTX-M15 and CTX-M55 represent the first molecular characterization of ESBL-producing E. coli isolates in Guatemala and it is important to understand the spread of these strains at the community environment. e ESBL-producing E. coli isolates showed high resistance to ciprofloxacin and trimethoprim sulfamethoxazole (78%) and low resistance levels for fosfomycin (2.5%) and nitrofurantoin (7.6%). e 11.39% of the strains showed resistance to a group of non-beta-lactam antibiotics. It is important to establish an active surveillance for these antibiotics in community strains because they are the first line treatment for strains producing ESBL

High clonal diversity of multidrug-resistant and extended spectrum beta-lactamase-producing Escherichia coli in a wastewater treatment plant.

Increasing beta-lactam resistance has led to the exploration of different places, such as wastewater treatment plants (WWTPs) which have been considered to be reservoirs and sources of bacterial resistance. This work aims to determine the presence of beta-lactamase-producing-Enterobacteriaceae in different points of a WWTP in Colombia. Six samplings were carried out in 2017 in the raw influent, aeration tanks, recycled sludge and final effluent of a WWTP. The beta-lactamase-producing-Enterobacteriaceae were detected and identified using phenotypic and molecular methods. Of the 353 isolates included, 28.3% corresponded to enterobacteria. The most frequent microorganisms were Escherichia coli (83%), Citrobacter freundii (11%) and Enterobacter cloacae complex (4%). The 97% of enterobacteriaceae had at least one beta-lactamase, and the most prevalent were the blaTEM (43.8%) and blaCTX-M-1group (35.8%) which were detected specially in recycled sludge and final effluent sample points. High percentage of multidrug resistance (to beta-lactams and non-beta-lactam antibiotics) was detected in E. coli (63.2%). Additionally, the typing by PFGE and MLST showed high genotypic diversity and the presence of the successful ST131 clone, globally spread. This work highlights the strong role of E. coli as a vector for the dissemination of resistance and the beta-lactamases in aquatic environments.

High frequency of aac(6')-Ib-cr gene associated with double mutations in gyrA and parC in Escherichia coli isolates from patients with urinary tract infections.

OBJECTIVES: The aims of this study were (i) to determine the frequency of plasmid-mediated resistance to fluoroquinolones (FQs) in Escherichia coli isolated from patients with urinary tract infections (UTIs) of nosocomial and community origin and (ii) to determine the relationships between the presence of extended-spectrum ß-lactamases (ESBLs), mutations in the gyrA and parC genes, and resistance to FQs. METHODS: A total of 71 E. coli isolates, including 38 ESBL-producers and 33 non-ESBL-producers, were analysed. The aac(6')-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6')-Ib-cr, a variant associated with FQ resistance. Detection of qnr genes was performed by multiplex PCR. In isolates that tested positive for these genes, the gyrA and parC genes were sequenced and the modulation factor of an efflux pump inhibitor was determined on the minimum inhibitory concentration (MIC) of norfloxacin. RESULTS: The frequencies of qnrS, qnrB and qnrA were 4.2%, 2.8% and 0%, respectively. The frequency of aac(6')-Ib-cr was 40.8% and this variant was associated with double mutations in gyrA and parC as well as resistance to FQs and ESBL production. Modulation of efflux pump activity was more frequent in resistant isolates that had a wild-type parC gene. CONCLUSION: An interplay of resistance mechanisms increased the level of resistance to FQs, and the high frequency of putative plasmid-mediated quinolone resistance genes associated with ESBL-producing isolates reduced therapeutic options to treat UTIs in the affected population.

[Rapid test for detection of susceptibility to cefotaxime in Enterobacteriaceae]. / Método rápido para la detección de la sensibilidad a cefotaxima en enterobacterias.

In this work an "in house" rapid test based on the change in pH that is due to hydrolysis for detecting Enterobacteriaceae susceptible to cefotaxime is evaluated. The strains of Enterobacteriaceae from 1947 urine cultures were assessed using MicroScan panels and the "in house" test. This rapid test includes red phenol solution and cefotaxime. Using MicroScan panels, 499 Enterobacteriaceae isolates were evaluated, which included 27 isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBL), 16 isolates of Klebsiella pneumoniae ESBL and 1 isolate of Klebsiella oxytoca ESBL. The "in house" test offers the following values: sensitivity 98% and specificity 97%, with negative predictive value 100% and positive predictive value 78%. The "in house" test based on the change of pH is useful in our area for detecting presumptively cefotaxime-resistant Enterobacteriaceae strains.

Antimicrobial resistance in Enterobacteriaceae in Brazil: focus on ß-lactams and polymyxins.

During the last 30 years there has been a dissemination of plasmid-mediated ß-lactamases in Enterobacteriaceae in Brazil. Extended spectrum ß-lactamases (ESBL) are widely disseminated in the hospital setting and are detected in a lower frequency in the community setting. Cefotaximases are the most frequently detected ESBL type and Klebsiella pneumoniae is the predominant species among ESBL producers. Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae became widely disseminated in Brazil during the last decade and KPC production is currently the most frequent resistance mechanism (96.2%) in carbapenem resistant K. pneumoniae. To date KPC-2 is the only variant reported in Brazil. Polymyxin B resistance in KPC-2-producing K. pneumoniae has come to an alarming rate of 27.1% in 2015 in São Paulo, the largest city in Brazil. New Delhi metallo-ß-lactamase was detected in Brazil in 2013, has been reported in different Brazilian states but are not widely disseminated. Antimicrobial resistance in Enterobacteriaceae in Brazil is a very serious problem that needs urgent actions which includes both more strict adherence to infection control measures and more judicious use of antimicrobials.

Faecal Escherichia coli isolates from healthy dogs harbour CTX-M-15 and CMY-2 ß-lactamases.

The presence of extended spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC ß-lactamase (pAmpC) producing Escherichia coli, along with the mechanisms of antimicrobial resistance and the molecular types of isolates, was investigated in faecal samples from 53 healthy dogs in Mexico. Samples were inoculated on Levine agar plates with 2 µg/mL cefotaxime for recovery of cefotaxime-resistant (CTX(R)) E. coli. CTX(R)E. coli isolates were recovered from 9/53 (17%) samples; one isolate was characterised from each positive sample. ESBL producing E. coli isolates were detected in 3/53 (6%) samples; these isolates carried the blaCTX-M-15 gene and one isolate also carried blaSHV-2. These three ESBL-positive E. coli isolates belonged to phylogroup A and sequence types ST617, ST410 or ST3944. The remaining 6/53 (11%) samples contained pAmpC positive isolates; these isolates carried the blaCMY-2 gene, which encodes CMY-2 ß-lactamase. These six isolates belonged to phylogroups A (n = 2), B1 (n = 1) and D (n = 3), and sequences types ST1431, ST57, ST93 and ST4565. One CMY-2 ß-lactamase positive E. coli isolate of lineage ST93 had the -32 mutation in the chromosomal ampC promoter/attenuator region. Five ESBL/pAmpC positive E. coli isolates carried class 1 integrons (dfrA17-aadA5, aadA and aadA/aadB arrays were detected in three isolates) and one isolate carried a class 2 integron (dfrA12-sat2-aadA1). The aac(6')Ib-cr, aac(3)-II, qnrB19, tet(A), tet(B), cmlA, and sul3 genes were also detected. All studied isolates showed unrelated PFGE-patterns. To our knowledge, this is the first description of ESBL-producing E. coli and the second of pAmpC-producing E. coli from healthy dogs in America. Our results suggest the potential zoonotic role of dogs in the transmission to humans of ESBL and pAmpC E. coli in the household environment.

Detection of multi drug resistant bacteria in major hospitals in Kano, North-West, Nigeria.

Two major hospitals in Kano, North West Nigeria have recorded increasing resistance of clinical pathogens to broad spectrum ß lactams, mediated by extended spectrum ß-lactamase (ESßL) and non ESBLs. A study was therefore undertaken to determine the occurrence and prevalence of plasmid and chromosomal mediated AmpC ßL and carbapenemase in addition to already known ESBL due to increasing resistance of pathogens from the two hospitals to carbapenems, cephamycins and flouroquinolones. Antibiogram tests and ESBL, AmpC and carbapenemase production tests were performed on all the isolates. AmpC and carbapenemase producers were further screened for AmpC inducibility and metallo beta lactamase production respectively. Majority of the isolates (> 80%) were resistant to both ß-lactam and non ß-lactam antibiotics. Reduced susceptibility to levofloxacin, nitrofurantoin, nalidixic acid and ofloxacin among the isolates were observed with the exception of P. aeruginosa which is totally resistant to imipenem and levofloxacin. An overall prevalence of 14.4%, 11.9% and 11.9.3% for ESßL, AmpC and carbapenemase was observed respectively. About 7.9% of the AmpC producers can over expressed the chromosomally mediated AmpC and 85.8% of the carbapenemase producers require metal for their action. Co-production of either of two and/or all of the enzymes was observed in E. coli, P. mirabilis and P. aeruginosa. Antibiotic resistance among isolates from the two hospitals is increasing and the major cause of this resistance in the pathogens studied are production of AmpC, carbapenemase (especially Metallo ß-lactamase) in addition to already known ESBL enzymes by the pathogens. Some of the isolates also possess the capacity to elaborate two or more of the enzymes concurrently, which would renders them resistant to a multitude of antibiotics.

Resistencia antimicrobiana en aislamientos clínicos de Klebsiella spp. y producción de B-lactamasas de espectro extendido en hospitales de Cuba / Antimicrobial resistance observed in clinical Klebsiella spp isolates and extended spectrum B lactamases in Cuban hospitals

Introducción: el género Klebsiella causa brotes hospitalarios, por cepas multidrogorresistentes en diferentes continentes y conlleva a un aumento en la morbimortalidad. Objetivos: identificar las especies de Klebsiella causantes de infecciones en hospitales cubanos, determinar la procedencia de los aislamientos según el servicio, y la susceptibilidad antimicrobiana, conocer la prevalencia de aislamientos productores de ß-lactamasas de espectro extendido (BLEE), y tipos, así como la susceptibilidad de los mismos a diferentes antimicrobianos de interés terapéutico. Métodos: se realizó un estudio descriptivo en 448 aislamientos de Klebsiella spp. recibidos en el Laboratorio Nacional de Referencia de Microbiología del IPK (LRNM/IPK) procedentes de 40 hospitales de 12 provincias del país durante el período de 2010 a 2012. La identificación de las especies se realizó mediante pruebas bioquímicas convencionales y por la técnica de Espectrometría de masas MS MALDI-TOF. Se determinó la susceptibilidad a 15 antimicrobianos mediante el método E-test y la producción de BLEE mediante el método de discos combinados según las recomendaciones del Instituto de Estándares Clínicos y de Laboratorio. Los genes blaESBL se determinaron mediante la reacción en cadena de la polimerasa según protocolo descrito previamente. Resultados: la especie prevalente fue Klebsiella pneumoniae (95,1 %), seguida por K. oxytoca (4,5 %) y K. ozaenae (0,4 %). Los aislamientos procedieron, principalmente, de los servicios de Unidades de Cuidados Intensivos (26,3 %), cirugía (22 %) y neonatología (13 %). La mayor resistencia se observó para las cefalosporinas (48-52 %), trimetoprim-sulfametoxazol (49 %), gentamicina (43 %), ácido nalidíxico (38 %) y tetraciclina (34 %). El 52 % de los aislamientos fueron productores de BLEE y prevaleció la enzima CTX-M (82 %) y la TEM (70 %). Conclusiones: se evidencia la repercusión clínica de Klebsiella spp. en hospitales cubanos con elevada resistencia a diferentes antimicrobianos. La producción de BLEE es un mecanismo de resistencia importante en esta bacteria en las que los carbapenémicos, la piperacilina-tazobactam, la colistina, y la tigeciclina juegan un rol terapéutico importante.

Introduction: the Klebsiella genus gives rise to many hospital outbreaks due to multi-drug-resistant strains on different continents and leads to increased morbidity and mortality. Objectives: to identify the Klebsiella species causing infections in Cuban hospitals, to determine the origin of isolates per service, their antimicrobial susceptibility and, to determine the production and type of extended-spectrum ß-lactamases (ESBLs) and the susceptibility of these isolations to several antimicrobials of therapeutic interest. Methods: a descriptive study was conducted on 448 Klebsiella spp. Isolates that were received in the national reference microbiology laboratory of "Pedro Kouri" Institute of Tropical Medicine from 40 hospitals located in 12 Cuban provinces during the 2010-2012 period. Species identification was based on conventional biochemical tests and mass spectrometry technique called MS MALDI-TOF. The susceptibility to 15 antimicrobials and the extended spectrum ß-lactamase production were determined by the E-test method and by the combined disks method, respectively, according to recommendations of the Institute of Clinical and Laboratory Standards. The polymerase chain reaction made it possible the detection of BlaESBL genes as indicated in the previously described protocol. Results: Klebsiella pneumoniae (95.4 %) was the prevalent species, followed by K. oxytoca (4.1%), and K. ozaenae (0.5 %). The isolates were mainly from the intensive care units (26.3 %), surgery (22 %), and neonatology (13%) services. The highest resistance rate was observed for cephalosporins (48-52 %), trimethoprim-sulfamethoxazole (49 %), gentamicin (43 %), nalidixic acid (38 %), and tetracycline (34 %). Fifty-two percent of the isolates were extended spectrum ß-lactamase producers, with CTX-M (82 %) and TEM (70 %) enzymes prevailing. Conclusions: This study shows the clinical impact of Klebsiella spp in Cuban hospitals, which is highly resistant to different antimicrobials. The production of extended spectrum ß-lactamases provides a significant resistance mechanism in Klebsiella in which carbapenems, piperacillin-tazobactam, cholistin and tigecycline play an important therapeutic role.

Diversidad de ß-lactamasas en aislamientos clínicos de enterobacterias / ß-lactamases diversity in clinical isolates of enterobacterias / Diversidade de ß-lactamases em isolamentos clinicos de enterobactérias

La rápida emergencia de resistencia a antimicrobianos debida a la presencia de b-lactamasas de espectro extendido (BLEE) tiene un impacto significativo en la salud pública. Las BLEEs son enzimas producidas por bacilos gramnegativos y confieren resistencia a las penicilinas, a todas las cefalosporinas y al aztreonam, pero no a los carbapenemes ni a las cefamicinas y la mayoría son inhibidas por el ácido clavulánico. El objetivo de este trabajo fue evaluar la resistencia a antibióticos b-lactámicos en aislamientos de Klebsiella pneumoniae, Escherichia coli y Proteus mirabilis y caracterizar las b-lactamasas responsables de dicha resistencia. Se analizaron 2.030 aislamientos (362 Klebsiella pneumoniae, 1.250 Escherichia coli y 175 Proteus mirabilis) provenientes de diferentes materiales clínicos de pacientes que concurrieron al Hospital Provincial del Centenario de la ciudad de Rosario (Santa Fe) durante el período 2008-2009. Los ensayos de sensibilidad antibiótica se realizaron de acuerdo con las recomendaciones del Clinical and Laboratory Standard Institute. Se confirmó la presencia de los genes codificantes de BLEE blaTEM, blaSHV, blaCTX-M y blaPER mediante la reacción en cadena de la polimerasa (PCR) utilizando cebadores específicos. Los aislados fueron caracterizados fenotípicamente como productores de BLEE y demostraron poseer varios genes bla. Se detectaron tres diferentes b-lactamasas BLEE derivadas de SHV, TEM y CTX-M y se demostró que pueden coexistir dos o más de estos genes en una misma bacteria.

The rapid emergence of antimicrobial resistance due to extended spectrum b-lactamases (ESBL) has a significant impact on public health. ESBL, produced by gram-negative bacilli, are enzymes that confer resistance to penicillins, cephalosporins and aztreonam, but not to carbapenems or cephamycins, and are usually inhibited by clavulanic acid. The aim of this study was to evaluate b-lactam resistance within isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis and to characterize the b-lactamases responsible for this resistance. A total of 2,030 strains (362 Klebsiella pneumoniae, 1,250 Escherichia coli, and 175 Proteus mirabilis) isolated from patients at Hospital Provincial del Centenario in Rosario-Santa Fe were analyzed from 2008 to 2009. Antibiotic sensitivity tests were performed according to Clinical and Laboratory Standard Institute recommendations. Molecular detection of ESBL-related bla genes, including blaTEM, blaSHV, blaCTX-M and blaPER was performed by polymerase chain reaction (PCR) using specific primers. The strains were phenotipically confirmed as ESBL producers and the isolates carried several bla genes. Three different b-lactamases were detected: SHV-related, TEM-related and CTX-M-related, showing that two or more genes may coexist in the same bacterium.

A rápida emergência de resistência a antimicrobianos devida à presença de b lactamases de espectro estendido (BLEE) tem um impacto significativo na saúde pública. As BLEEs são enzimas produzidas por bacilos gram-negativos e conferem resistência às penicilinas, a todas as cefalosporinas e ao aztreonam, mas não aos carbapenêmicos nem às cefamicinas e a maioria são inibidas pelo ácido clavulânico. O objetivo deste trabalho foi avaliar a resistência a antibióticos b-lactâmicos em isolamentos de Klebsiella pneumoniae, Escherichia coli e Proteus mirabilis e caracterizar as b-lactamases responsáveis por tal resistência. Foram analisados 2.030 isolamentos (362 Klebsiella pneumoniae, 1.250 Escherichia coli e 175 Proteus mirabilis) provenientes de diferentes materiais clínicos de pacientes que foram ao Hospital Provincial do Centenário da cidade de Rosario (Santa Fe) durante o período 2008-2009. Os ensaios de sensibilidade antibiótica foram realizados de acordo com as recomendações do Clinical and Laboratory Standard Institute. Confirmou-se a presença dos genes codificantes de BLEE blaTEM, blaSHV, blaCTX-M e blaPER mediante a reação em cadeia da polimerase (PCR) utilizando cevadores específicos. Os isolados foram caracterizados fenotipicamente como produtores de BLEE e demonstraram possuir vários genes bla. Foram detectadas três diferentes b-lactamases derivadas de SHV, TEM e CTX-M e se demonstrou que podem coexistir dois ou mais destes genes numa mesma bactéria.

Diversidad de ß-lactamasas en aislamientos clínicos de enterobacterias / ß-lactamases diversity in clinical isolates of enterobacterias / Diversidade de ß-lactamases em isolamentos clinicos de enterobactérias

La rápida emergencia de resistencia a antimicrobianos debida a la presencia de b-lactamasas de espectro extendido (BLEE) tiene un impacto significativo en la salud pública. Las BLEEs son enzimas producidas por bacilos gramnegativos y confieren resistencia a las penicilinas, a todas las cefalosporinas y al aztreonam, pero no a los carbapenemes ni a las cefamicinas y la mayoría son inhibidas por el ácido clavulánico. El objetivo de este trabajo fue evaluar la resistencia a antibióticos b-lactámicos en aislamientos de Klebsiella pneumoniae, Escherichia coli y Proteus mirabilis y caracterizar las b-lactamasas responsables de dicha resistencia. Se analizaron 2.030 aislamientos (362 Klebsiella pneumoniae, 1.250 Escherichia coli y 175 Proteus mirabilis) provenientes de diferentes materiales clínicos de pacientes que concurrieron al Hospital Provincial del Centenario de la ciudad de Rosario (Santa Fe) durante el período 2008-2009. Los ensayos de sensibilidad antibiótica se realizaron de acuerdo con las recomendaciones del Clinical and Laboratory Standard Institute. Se confirmó la presencia de los genes codificantes de BLEE blaTEM, blaSHV, blaCTX-M y blaPER mediante la reacción en cadena de la polimerasa (PCR) utilizando cebadores específicos. Los aislados fueron caracterizados fenotípicamente como productores de BLEE y demostraron poseer varios genes bla. Se detectaron tres diferentes b-lactamasas BLEE derivadas de SHV, TEM y CTX-M y se demostró que pueden coexistir dos o más de estos genes en una misma bacteria.(AU)

The rapid emergence of antimicrobial resistance due to extended spectrum b-lactamases (ESBL) has a significant impact on public health. ESBL, produced by gram-negative bacilli, are enzymes that confer resistance to penicillins, cephalosporins and aztreonam, but not to carbapenems or cephamycins, and are usually inhibited by clavulanic acid. The aim of this study was to evaluate b-lactam resistance within isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis and to characterize the b-lactamases responsible for this resistance. A total of 2,030 strains (362 Klebsiella pneumoniae, 1,250 Escherichia coli, and 175 Proteus mirabilis) isolated from patients at Hospital Provincial del Centenario in Rosario-Santa Fe were analyzed from 2008 to 2009. Antibiotic sensitivity tests were performed according to Clinical and Laboratory Standard Institute recommendations. Molecular detection of ESBL-related bla genes, including blaTEM, blaSHV, blaCTX-M and blaPER was performed by polymerase chain reaction (PCR) using specific primers. The strains were phenotipically confirmed as ESBL producers and the isolates carried several bla genes. Three different b-lactamases were detected: SHV-related, TEM-related and CTX-M-related, showing that two or more genes may coexist in the same bacterium.(AU)

A rápida emergÛncia de resistÛncia a antimicrobianos devida O presenþa de b lactamases de espectro estendido (BLEE) tem um impacto significativo na saúde pública. As BLEEs sÒo enzimas produzidas por bacilos gram-negativos e conferem resistÛncia Os penicilinas, a todas as cefalosporinas e ao aztreonam, mas nÒo aos carbapenÛmicos nem Os cefamicinas e a maioria sÒo inibidas pelo ácido clavulÔnico. O objetivo deste trabalho foi avaliar a resistÛncia a antibióticos b-lactÔmicos em isolamentos de Klebsiella pneumoniae, Escherichia coli e Proteus mirabilis e caracterizar as b-lactamases responsáveis por tal resistÛncia. Foram analisados 2.030 isolamentos (362 Klebsiella pneumoniae, 1.250 Escherichia coli e 175 Proteus mirabilis) provenientes de diferentes materiais clínicos de pacientes que foram ao Hospital Provincial do Centenário da cidade de Rosario (Santa Fe) durante o período 2008-2009. Os ensaios de sensibilidade antibiótica foram realizados de acordo com as recomendaþ§es do Clinical and Laboratory Standard Institute. Confirmou-se a presenþa dos genes codificantes de BLEE blaTEM, blaSHV, blaCTX-M e blaPER mediante a reaþÒo em cadeia da polimerase (PCR) utilizando cevadores específicos. Os isolados foram caracterizados fenotipicamente como produtores de BLEE e demonstraram possuir vários genes bla. Foram detectadas trÛs diferentes b-lactamases derivadas de SHV, TEM e CTX-M e se demonstrou que podem coexistir dois ou mais destes genes numa mesma bactéria.(AU)

Detection of extended spectrum beta lactamases, ampc beta lactamases and metallobetalactamases in clinical isolates of ceftazidime resistant Pseudomonas Aeruginosa.

We studied the prevalence of ceftazidime resistance in Pseudomonas aeruginosa and the rates of extended-spectrum ß-lactamase (ESBL), AmpC ß-lactamase (AmpC) and metallo-ß-lactamase (MBL) production among the ceftazidime resistant Pseudomonas aeruginosa. A very high rate of MBL production was observed, which suggested it to be an important contributing factor for ceftazidime resistance among Pseudomonas aeruginosa.