Phenotypic Demonstration of ß-lactamase (ESßLs, MßLs, and Amp-C) among MDR Pseudomonas aeruginosa isolates obtained From Burn wound infected in Yemen

In 2017, the World Health Organization published its first-ever list of antimicrobial-resistant bacteria “prioritypathogens,” a catalog of 12 families of bacteria posing the greatest threat to human health. This list focuses onthe risk of Gram-negative bacteria for multiple drug-resistant. Pseudomonas aeruginosa was at the top of the listand critical. A current study aiming to demonstrate the prevalence of β-lactamase among multidrug-resistant P.aeruginosa strains isolated from burn wound patients phenotypically. The isolates were identified then antibioticsusceptibility tested against 10 antipseudomonal agents, finally, phenotypically β-lactamase (ESβLs, MβLs, andAmp-C) production screened by combined disk diffusion test and Imipenem-ethylenediaminetetraacetic acid.Results in the current study identified 98 P. aeruginosa isolates from 200 clinical specimens obtained from burnwound patients. Our result showed 65 (66.3%) of the 98 P. aeruginosa isolates were multiple drug-resistant(MDR) strains. Out of 65 isolates, 37 (56.9%), 21 (32.3%), and 40 (61.5%) were ESβLs, MβLs, and Amp-Cproducing P. aeruginosa, respectively, according to phenotypic detection method. We found co-expression ofvarious β-lactamases. In the present study, 16 isolates showed co-existence of AmpC + ESBL, 16 isolates werehaving ESBL + MBL + AmpC, and five isolates were having co-existence of ESBL + MBL. The occurrence ofESβLs, MβLs, and Amp-C producing P. aeruginosa was demonstrated, calling for phenotypical determinationof antibiotic resistance mechanisms should be performed regularly to guide antibiotic selection during therapy.Significant conclusions drawn from this work include a rise in the rate of β-lactamase (ESβLs, MβLs, andAmp-C) in MDR P. aeruginosa. Later research should, therefore, focus on the study of molecular characterization.

Phenotypic & genotypic profile of antimicrobial resistance in Pseudomonas species in hospitalized patients

Background & objectives: Nosocomial infections caused by multidrug-resistant, Pseudomonas species have become a major clinical and public health concern. The aim of this study was to characterize phenotypic and genotypic profile of antimicrobial resistance (AMR) in Pseudomonas spp. isolated from hospitalized patients. Methods: A total of 126 consecutive, non-duplicate isolates of Pseudomonas spp. isolated from various clinical samples were included in the study over a period of two years. Identification and antimicrobial sensitivity was performed using automated culture system according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Phenotypic detection of extended-spectrum ?-lactamases (ESBLs), Amp-C ?-lactamase (AmpC) and metallo-?-lactamases (MBLs) were done by various combinations of disc-diffusion and E-test methods, followed by polymerase chain reaction-based detection of ?-lactamase-encoding genes. Results: Among 126 clinical isolates, 121 (96.1%) isolates were identified as Pseudomonas aeruginosa. Most of the isolates were recovered from pus sample, 35 (27.8%) followed by urine, 25 (19.84%); endotracheal aspirate, 24 (19.04%); blood, 14 (11.11%) and sputum, four (3.17%). The highest rate of resistance was against ticarcillin-clavulanic acid, 113 (89.7%) followed by meropenem, 92 (72.5%) and ceftazidime, 91 (72.3%). Overall, ESBLs, AmpC and carbapenemase production was detected in 109 (96.4%), 64 (50.8%) and 105 (94.6%) isolates by phenotypic methods. The most prevalent ESBL gene was blaTEMin 72 (57.1%) and the least prevalent was blaSHVin 19 (15.1%) isolates. AmpC gene was seen less compared to ESBL gene. The most prevalent carbapenemases gene was blaNDM-141 (46.06%) followed by blaVIM and blaOXA-1. Interpretation & conclusions: Our findings suggested that a high rate of ESBLs and carbapenemases production was observed in Pseudomonas spp. Therefore, phenotypic and genotypic detection of AMR needs to be combined for better characterization of resistance patterns in Pseudomonas spp.

Identification Of Non– Fermenter Gram Negative Bacilli (NFGNB) From Various Clinical Isolates With Special Reference To Carbapenem Resistance In Various Isolates.

Non Fermenter Gram negative bacilli (NFGNB) has emerged as important hospital pathogens they are more significant as they are found to be multi drug resistant. Resistance to carbapenems is common among NFGNB. AIMS & OBJECTIVES: To isolate & identify NFGNB from various clinical samples and to detect resistance to carbapenem in isolates resistant to Imipenem. MATERIAL & METHOD: NFGNB isolated from various samples were speciated using standard tests. Isolates resistant to Imipenem were subjected to detection of MBLs(Metallo-β-lactmase) and Amp-C. RESULTS: Out of 1566 samples received, NFGNB were 200. Among them 112 were Pseudomonas aeruginosa from which 31 were found to be resistant to Imipenem, of which 3 were MBLproducer by Modified Hodge test while 4 were MBLproducer by EDTAdisc synergy test. Out of 200 NFGNB 71 were Acinetobacter baumanii, of which 23 were found to be resistant to Imipenem, of which 6 were MBLproducer by Modified Hodge test, while 4 were seen to be MBL producer by EDTAdisc synergy test. Nineteen isolates of Acinetobacter baumanii were found to be resistant to cefoxitin of which 6 were found to be Amp-C producer by Amp-c disc test. None of the Pseudomonas aeruginosa were Amp-C producer. Other NFGNB isolated were either sensitive to Imipenem or if resistant were not MBLor Amp-C producer.

Coexpression of ESBL, Amp C and MBL in gram negative bacilli.

Background: Resistant bacteria are emerging worldwide as a threat to the favourable outcome of common infections in community and hospital settings. Extended Spectrum Beta-Lactamases (ESBLs), AmpC β lactamases and Metallo-β Lactamases (MBL) are the three important mechanism of resistance to beta lactam drugs in the bacteria. The objective of the study was to screen gram negative isolates for co-expression of extended spectrum β-lactamase, Amp C β-lactamase and Metallo β-lactamase production. Methods: In this study 50 (27 male & 23 female) adult skulls were investigated to determine the type of asterion, its distance from important bony landmarks and also the nearby venous sinuses were measured. Results: Seven hundred and six isolates from various clinical samples from Kamineni institute of medical sciences Hospital, Narketpally, were processed during the period of October 2010 to September 2012. Gram negative bacilli were identified by colony morphology, gram stain, motility, enzyme detection tests, etc. ESBL detection was carried but by two procedures like double disc synergy tests (DDST) and phenotypic confirmatory disc diffusion test (PCDDT). AmpC Beta-lactamase detection was done by AmpC Disc Test. MBL production was tested by Imipenem-EDTA combined disc test. Conclusions: Klebsiella was the commonest isolate (28.47%) followed by E coli (26.48%), Pseudomonas aeruginosa (19.54%), Enterobacter (8.92%), Acinetobacter (8.92%) and Citrobacter (7.64%). A total of 272 out of 706 gram negative isolates were ESBL producers. ESBL production was seen more in E. coli followed by Klebsiella and P. aeruginosa. A total of 73 out of 706 isolates were inducible Amp C producers. AmpC production was seen more in Acinetobacter. A total of 65 out of 706 isolates were MBL producers. MBL Production was seen more in E. coli.

Phenotypic detection of Extended Spectrum Beta-Lactamases and Amp C Beta- Lactamases among nosocomial isolates in a tertiary care hospital.

Background & objectives: Extended-Spectrum Beta-Lactamases (ESBLs) is an important resistance mechanism in Enterobacteriaceae infections. Lack of standard guidelines from Clinical Laboratory Standards Institute (CLSI) for Amp C beta-lactamase detection poses a problem. This study was undertaken to detect ESBLs by phenotypic tests and Amp C beta-lactamase by inhibitor based method. Material and Methods: 200 consecutive non-repetitive isolates of E.coli, Klebsiella and Proteus from clinical samples were screened for ESBLs as per CLSI guidelines and confirmed by PCDT, DDST and E-tests (AB Biodisk, Biomerieux). Amp C beta lactamases were screened by cefoxitin resistance and confirmed by inhibitor (Cloxacillin) based method. Simultaneous occurrence of Amp C and ESBLs was detected by combined disk test (Neo-Sensitabs and Diatabs). Descriptive and Kappa statistics were used. Results: Out of 200 isolates studied, 131 were initially screened as ESBL producers and later 114 (57%) were confirmed by phenotypic methods. E-Test was found most sensitive phenotypic test as compared to PCDT and DDST. 13 strains resistant to cefoxitin (30μg) were found to be pure Amp C producers. Combined disk test detected 36 to be ESBL and Amp C co-producers. Surprisingly, six isolates found sensitive to cefoxitin disk were confirmed as Amp C producers by cloxacillin disk inhibition test. Conclusion: 57% ESBLs and 27.5% Amp C producers were isolated from nosocomial pathogens showing significant resistance to 3rd generation cephalosporins. Phenotypic confirmation by E-test, PCDT & DDST were useful for ESBL identification and for detection of Amp C, cloxacillin was found to be an effective inhibitor.

Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae.

Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR). Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25) and K. pneumoniae (n = 52) were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL). Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test). Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii), DHA (Dhahran Hospital, Saudi Arabia), ACC (Ambler class C), EBC (Amp C origin - Enterobacter cloacae) groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

Phenotypic & molecular characterization of AmpC β-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

Background & objectives: AmpC β-lactamases which are often plasmid mediated hydrolyze all β-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC β-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. Methods: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. Results: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n=25), EBC (n=10), FOX (n = 4), CIT (n=3), EBC-ACC (n=2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. Interpretation & Conclusions: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.

Prevalência de Pseudomonas aeruginosa produtoras de β lactamases do tipo AMP-C em isolados clínicos de Santa Maria - RS / Prevalence of Pseudomonas aeruginosa AMP-C β lactamases in clinical isolates of Santa Maria - RS

Microbiologia proporcionar informações claras ao clínico, reportando corretamente os resultados, para que o tratamento seja eficaz e o mais seguro possível, evitando-se assim, a falha terapêutica e a resistência microbiana. As β-lactamases são enzimas que catalizam a hidrólise de ligações carbono-nitrogênio, separando a base do substrato inativando os antimicrobianos β-lactâmicos acarretando em falha terapêutica. Neste trabalho analisou-se 90 amostras de Pseudomonas aeruginosa provenientes de isolados clínicos de Santa Maria-RS. Empregou-se a técnica de disco difusão (Kirby-Bauer), com aproximação dos discos de Cefoxitina e Cefotaxima, onde31 cepas (34,4%) apresentaram positividade in vitro para a produção deβ-lactamases do tipo Amp-C. As cepas do grupo CESP (Citrobacter freundii, Enterobacter sp., Providencia sp., Serratia marcescens) apresentam resistência à Cefoxitina no antibiograma e não necessitariam de testes especiais para a detecção, uma vez que a enzima é produzida de forma constitutiva. Porém, alguns isolados de Pseudomonas aeruginosa, devido à produção de Amp-C em pequenas quantidades, podem apresentar sensibilidade in vitro à Cefoxitina, mas in vivo este antimicrobiano não terá ação ou sua ação será reduzida, ocasionando falha terapêutica.

Producción de ß-lactamasas de espectro extendido (BLEE) en cepas de Acinetobacter baumannii aisladas en hospitales de la VIIIª Región, Chile / Extended spectrum ß-lactamases (ESBL) production in Acinetobacter baumannii strains isolated from Chilean hospitals belonging to VIII Region

The resistance of Acinetobacter baumannii to ß-lactam antibiotics is mainly due to the synthesis of ß-lactamases. From a clinical point of view, this bacteria and others, grouped under the acronym SPACE (S: Serratia, P: Pseudomonas, A: Acinetobacter, C: Citrobacter, E: Enterobacter) are essentially Amp-C ß-lactamases producers. There is no local information about ESBL presence in Acinetobacter. We studied ESBL production using the Ho and col. technique modified by adding cloxacillin as chromosomal ß-lactamases inhibitor. From 69 isolates, with resistance to at least one third generation cephalosporin, only 7 showed positive synergy test. Four of these amplified for TEM family gene, and one of these amplified also for the OXA family. Our study found a low ESBL production percentage, which agrees with the premise of Amp-C as the main mechanism of resistance to ß-lactam antibiotics in A. baumannii. However, the ESBL description in these bacteria emphasizes the capacity of expressing multiple resistance mechanisms.

La resistencia de Acinetobacter baumannii a antimicrobianos ß-lactámicos se debe fundamentalmente a la síntesis de ß-lactamasas. Del punto de vista clínico se considera que esta bacteria, y otras agrupadas en el acrónimo SPACE (Serratia, Pseudomonas, Acinetobacter, Citrobacter, Enterobacter), son predominantemente productoras de ß-lactamasas tipo AmpC. No hay información en nuestro país sobre presencia de ß-lactamasas de espectro extendido (BLEE) en Acinetobacter. Se estudió la producción de BLEE en cepas de Acinetobacter, mediante una modificación de la técnica de Ho y col adicionando cloxacilina como inhibidor de ß-lactamasas cromosomales. De 69 cepas con resistencia al menos a una cefalosporina de tercera generación, sólo siete presentaron sinergia positiva. Cuatro cepas amplificaron por RPC un fragmento intragénico de genes de familia TEM y una de ellas amplificó, además, para el gen de la familia OXA. Se evidenció un bajo porcentaje de producción de BLEE, lo que confirma que la producción de Amp-C es el principal mecanismo de resistencia de A. baumannii a ß-lactámicos. Sin embargo, la descripción de BLEE en esta bacteria, enfatiza su capacidad de albergar múltiples mecanismos de resistencia.

Rapid detection of Amp C ?-Iactamase in Enterobacter cloacae by PCR / 第二军医大学学报

Objective:To establish a new method to detect Amp C?-lactamase by PCR. Methods:The amp C and amp D gene fragments of E. cloacae were amplified by the amp C and amp D primers to detect Amp C ?-lactamase, especially the enduring and highly productive enzymes. Results:Totally 193 of 214 strains of E. cloacae were positive for amp C gene , implicating most strains of E. cloacae had the ability to produce the enzyme. Sixteen of the 193 strains (amp C positive) were negative for amp D genes, implicating these 16 strains could produce the enduring and highly productive enzymes. The results were confirmed by cefoxitin three-dimensional test. Conclusion:The new method to detect Amp C ?-lactamase, especially the enduring and highly productive enzymes,by amp C and amp D primers is a rapid and accurate method.