Phenotypic Confirmatory Method Research Articles

Fosfomycin susceptibility among ESBL producing gram negative bacilli causing urinary tract infections

Urinary tract infection is one of the common infection encountered in day to day practice. Due to emergence of drug resitance among uropathogens treatment options have become limited. Fosfomycin being a safe oral antibiotic is being used widely to treat multidrug resistant uropathogens. In the present study 831 (48.45%) samples that yielded significant growth were processed out of 1715 sample for ESBL detection by double disc synergy and phenotypic confirmatory method. E.coli constituted the predominant isolate (60.4%) followed by K.pneumoniae. 256 (30.80%) samples yielding growth were from out patients and 575 from inpatients. Over all 44% of isolates in the present study were ESBL producers. 50% of Ecoli were ESBL producers. 70.64% of ESBL isolates were susceptible to fosfomycin in vitro. Present study finding suggest that resistance to fosfomysin is on rise even though majority of ESBLs were sensitive to it. The current study recommends to use fosfomycin only after testing susceptibility among uropathogens.

Phenotypic methods for detection of Amp C b lactamases in Gram negative clinical isolates of a tertiary care hospital

: ESBLs (Extended spectrum beta lactamases) are detected routinely in most laboratories but Amp c Beta lactamases are neglected to large extent. Pseudosusceptibility seen in Amp C producers leads to resistance to extended-spectrum cephalosporins resulting in inappropriate antimicrobial regimens and therapeutic failure. Thus, there is a need to know an appropriate phenotypic method for easy detection of Amp C β-lactamases as they have clinical relevance.: To detect and compare different phenotypic methods for Amp C β-lactamases in Gram-negative clinical isolates of Enterobacteriaceae familyA prospective study was done at Mamata Medical college, Khammam for period of three months. Phenyl boronic acid method, Cefoxitin Cloxacillin-Double disc synergy test, TRIS EDTA method, Disc approximation test were done on isolates after screening by Cefoxitin disc.: Out of 140 isolates tested, 80(57%) were positive (resistant) for screening test by cefoxitin. Out of them 61(76.2%) were Escherichia coli, 16 (20.1%) isolates were Klebsiella pneumoniae and 3 (3.75%) were Enterobacter sp. Phenotypic confirmatory methods by Cefoxitin Cloxacillin Double disc Synergy (CC-DDS) test showed zone difference of >4mm in 38(47.5%) isolates , by Phenylboronic acid method (PBA) > 5mm zone difference was observed in 34(42.5%) isolates, by TRIS EDTA method 32 (40%) showed indentation near EDTA disc and by Disc approximation test 32(40%) were positive for Amp C production. E coli was the commonest isolate showing Amp c production by all four methods.: CC-DDS method has better detection rate compared to other phenotypic confirmatory methods. We suggest CC-DDS method as it is easy to perform.

Expressed and Silent Carbapenemase Genes Detected by Multiplex PCR in both Carbapenem-Resistant and Phenotypically-Susceptible Gram Negative Bacilli

ABSTRACT Introduction Carbapenemase-producing Gram-negative bacilli have been major culprits in hospital-associated infections (HAIs), particularly in critically ill patients suffering device-associated infections (DAIs). The current study aimed to investigate the performance of the modified Hodge test (MHT) as a phenotypic confirmatory method for the detection of carbapenemase-producing Gram-negative bacilli and to compare it to the gold standard PCR for the detection of carbapenemase production in both non-susceptible and phenotypically susceptible isolates. The latter were expected to harbor silent carbapenemase genes, as suspected from the inappropriate response to carbapenem therapy. Methods Ninety-five bacterial isolates from 75 critically ill patients were collected over 6 months from several ICUs at Cairo University Hospitals. The isolates were subjected to antibiotic susceptibility testing (AST) for carbapenems and were further screened by MHT, followed by genotypic analysis via multiplex PCR. Results Enterobacteriaceae were the most commonly isolated pathogens (55.8% of the total isolates), followed by Acinetobacter spp. (24%). Lower respiratory tract infections were the most common HAIs (42.11%), followed by surgical site infections (27.37%). All isolates demonstrating carbapenem resistance by AST were found to harbor at least one of the following carbapenemase genes: blaKPC, blaOXA-48, blaIPM, blaVIM, and blaNDM-1 . Alarmingly, 97.8% of the isolates which exhibited carbapenem-susceptible profile and negative MHT were harboring carbapenemase genes as confirmed by multiplex PCR. With the exception of one isolate (E. coli) which was not harboring any carbapenemase gene, the remaining 94 bacterial isolates were found to carry either a single or multiple carbapenemase genes. Conclusion The silent dissemination of different classes of carbapenemases even in isolates with negative MHT is a daunting challenge. It necessitates the implementation of strict antibiotic stewardship along with updated and actionable approach to detect non-expressed carbapenemase genes in phenotypically susceptible isolates.

Limitation of Double Disc Diffusion Method as a phenotypic confirmatory method for ESBL producing organisms in Clinical Microbiology Laboratory

Limitation of Double Disc Diffusion Method as a phenotypic confirmatory method for ESBL producing organisms in Clinical Microbiology Laboratory

Molecular testing of Klebsiella pneumoniae contaminating tissue allografts recovered from deceased donors.

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9-2.3) (P value<0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.

Prevalence of extended spectrum ß-Lactamase and Carbapenemase producing isolates of Klebsiella SPP in a tertiary care hospital

OBJECTIVES: Wide spread use of s lactam antibiotics has lead to resistance to these group of antibiotics in bacterial pathogens due to various types of s lactamase production. Klebsiella pneumoniae harboring Extended spectrum s lactamase (ESBL)) and carbapenemases are a serious problem worldwide. Klebsiella pneumoniae is an established pathogen responsible for multidrug resistant hospital acquired infections. Our study was undertaken to detect prevalence of ESBL and KPC producing Klebsiella spp in a tertiary care hospital and to check their antibiotic resistance pattern by phenotypic methods recommended by CLSI. METHODS: A total of 120 non enteric clinical isolates klebsiella spp were speciated and antibiotic sensitivity was determined by Kirby Bauer disc diffusion method. Isolates found to be screen positive for possible ESBL and carbapenemase production were further confirmed by phenotypic confirmatory methods recommended by CLSI. RESULTS: I09 isolates were found to be screen positive for ESBL, out of which 29 isolates (27%) were confrmed ESBL producer by combined disc method. A total of 11 isolates were found to be screen positive for carbapenemase production out of which 4 (36%) were confirmed carbapenemase (KPC) producer by Modified Hodge test (MHT). All the ESBL and carbapenemase producing Klebsiella isolates were found to be multidrug resistant (MDR). 93% (27/29) ESBL positive isolates were sensitive to imipenem. ESBL producers also showed coresistance to amikacin 52% (15/29), ciprofloxacin 41% (12/29).Two carbapenemase producing 2 isolates were highly resistant and found to be sensitive to only colistin and tigecycline. CONCLUSION: Failure to detect MDR organisms like ESBL and KPC producers on time may lead to outbreak of hospital acquired infections by such strains. So clinical laboratories should routinely do such easy to perform screening tests to detect these resistance mechanisms.

Evaluation of Nordmann, Dortet, and Poirel test for the identification of extended spectrum betalactamase production among urinary isolates of Escherichia coli.

CONTEXT:Current phenotypic techniques for extended spectrum beta lactamase (ESBL) detection can be interpreted after 24 h of incubation only, resulting in a delay in initiating therapy. Nordmann, Dortet, and Poirel (NDP) in 2012 proposed a novel test named ESBL NDP to overcome this limitation.AIMS:This study aimed to evaluate the ESBL NDP test for the identification of ESBL among Escherichia coli isolates against the Clinical Laboratory Standards Institute-recommended phenotypic confirmatory method.SETTINGS AND DESIGN:This cross-sectional study was conducted over a period of 3 months on a sample size of 100.SUBJECTS AND METHODS:One hundred nonduplicate clinically significant E. coli urinary isolates positive by initial screening test for ESBL were subjected to the ESBL NDP test and phenotypic confirmatory test. The NDP test was evaluated by determining the sensitivity, specificity, kappa value, and confidence interval (CI) for kappa.RESULTS:The phenotypic confirmatory test and the ESBL NDP test were positive in 82% and 63% of the isolates, respectively. ESBL NDP test had a sensitivity and specificity of 76% and 100%, positive and negative predictive values of 100% and 48%, respectively, kappa value of 0.54 (moderate agreement), and 95% CI for kappa of 0.43–0.66. The time to positivity was 1 h in 93.6% of the isolates.CONCLUSION:The NDP test showed a good specificity, with time to positivity of 1 h. The low sensitivity could be due to the difference in the phenotypic type of ESBL producer and technical reasons.

ESBL producing Pseudomonas aeruginosa in clinical specimens: Is it a scary nightmare or paper tiger?

Background: Pseudomonas aeruginosa well known as opportunistic pathogen, has been implicated in life threatening nosocomial infections in recent years. This can be attributable to acquired resistance by plasmid mediated different types of extended spectrum beta lactamases(ESBL). The present study was undertaken to detect the ESBL production of Pseudomonas aeruginosastrains and analyze their susceptibility pattern. Methods: Various clinical specimens received in our laboratory were processed and Pseudomonas aeruginosa was identified as per standard microbiological procedure. A total of 132 clinical isolates of Pseudomonas aeruginosa identified during the study period were included in the study. All isolates were subjected for ESBL screening test. Potential ESBL producer was then subjected for ESBL Phenotypic confirmatory test –Disc Diffusion method(PCDDT). Antimicrobial susceptibility test was performed by Kirby – Bauer disc diffusion method on all confirmed isolates by PCDDT method as per Clinical Laboratory Standard Institute (CLSI 2016) guidelines. Results: By phenotypic confirmatory method, the frequency of occurrence of ESBL among the isolates identified was 21.96% (n = 29).The highest number of ESBL producers was obtained from urine samples(27.7%) followed by respiratory infection (23.68%) and wound infection (22.95%). In-vitro susceptibility of ESBL producers revealed high level of resistance to third generation cephalosporin. Low resistance was observed to Imipenem (3.4%) and Piperacillin – Tazobactam (13.79%). Conclusion: Our study highlights the unique challenge imposed by Pseudomonas aeruginosa as the therapeutic choices are limited by ESBL production. To overcome this issue, we recommend routine ESBL detection and surveillance of antibiotic resistance in hospital settings. Keywords: Pseudomonas aeruginosa, ESBL, Third generation cephalosporins, Imipenem, Urine, ESBL Screening test

Comparison of Two Phenotypic Double Disc Diffusion Test (DDDT) for Detection of Extended Spectrum β-Lactamase Production by Enterobacteriaceae and Pseudomonas Species at a Tertiary Care Hospital in Bangladesh

Background: The detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is of importance for infection control and epidemiological surveillance. Objective: The purpose of the present study was to compare two phenotypic methods for the detection of ESBL-positive Enterobacteriaceae and Pseudomonas species. Methodology: This cross sectional study was carried out in the Department of Microbiology at Mymensingh Medical College, Bangladesh from January 2011 to June 2011. Patients of all ages and genders presented with UTI or wound infection were taken as study population. Gram negative bacilli (GNB) were analyzed by two methods used for routine susceptibility testing which were Disk diffusion methods and MIC reduction methods. Two methods designed for the detection of ESBL production Ceftazidime and Ceftazidime plus Clavulinic acid, (CAZ/CAZC) and Cefotaxime and Cefotaxime plus Clavulinic Acid (CTX/CTXC) were used and the PCR was considered as gold standard for evaluation of the other test methods. Result: A total number of 300 GNB were isolated and identified of which 214(71%) were ESBL positive. For the disk diffusion method, resistant to third generation Cephalosporins were the highest 87.0%, when tested by ceftazidime and by MIC reduction methods were 67%. For the phenotypic confirmatory methods, specificities were 64% by (C AZ/CAZC), 59% by (CTX/CTXC) and by both method 52%. Among the phenotypic confirmatory ESBL positive strains by Genotypic method ESBL positive were 50.46% TEM, 18.69 % SHV and 46.72% CTX-M gene. Conclusion: Two-step strategies using both DDDT phenotypic methods are useful diagnostic tools for the detection of ESBL from the Gram negative bacilli.

Detection of Extended Spectrum Beta-Lactamase Producing Escherichia Coli on Water at Hafr Al Batin, Saudi Arabia

In this study antibiotic resistant Escherichia coli from household water collected in Hafr AlBatin, Saudi Arabia, were characterized by prevalence of extended spectrum betalactamase (ESBL). Samples were collected from drinking and washing water at 12 locations. From the 144 samples obtained, Millipore membrane filters incubated on trypton soya agar plates produced colonies that yielded 34 isolates of E. coli as verified by biochemical tests. Isolates suspected ESBL producing were tested by using MicroScan analysis and disc diffusion test as ESBL phenotypic confirmatory methods. Phenotypically confirmed ESBL isolates were examined for antimicrobial susceptibility against 30 antibiotics and amplification of blaVEM, blaCTX, blaTEM, blaGES and blaSHV genes by polymerase chain reaction. Out of 34 E. coli isolates, only 6 (17.6%) were positive for ESBL producing according to MicroScan analysis. Disk diffusion as a confirmatory test indicates sensitivity of MicroScan system. PCR results indicated that the VEB was the most prevalent (83.3%) followed by CTX gene (16.6%) between these isolates. The ESBL-producing E. coli isolates was fully susceptible to pip/tazo (100%) and fully resistance to ampicillin, cefazolin and pepracillin (100%). This study showed that ESBL-producing E. coli are multidrug-resistant and existent in Hafr Al Batin’s water. Also, data indicated that wastewater maybe contributes as a source and reservoir of antibiotic resistance.

English

Extended spectrum beta lactamase (ESBL) producing members of the family Enterobacteriaceae are a major health problem in the hospitals and community. These enzymes confer bacterial resistance to penicillins, first, second and third generation cephalosporins and aztreonam. The prevalence of ESBLs varies from organism to organism and is increasing day by day. The study was designed to test typhoidal salmonellae for production of ESBL. One hundred and fifty eight (158) isolates of typhoidal salmonellae; Salmonella Typhi (n=126), Salmonella Paratyphi A (n=26) and Salmonella Paratyphi B (n=6) were collected from different hospitals of Lahore and Gujranwala. The isolates were identified morphologically, biochemically (API-20E) and serologically (BD Difco, USA). ESBL production has been tested by three methods; CLSI screening method, double disk diffusion synergistic method and CLSI phenotypic confirmatory method. CLSI screening method detected twenty two (22) strains as ESBL producers. However, when tested by the CLSI confirmatory method and by disk diffusion synergistic method, none proved to be ESBL producer. Based on our study, we concluded that the extended spectrum beta lactamase enzyme does not exist in tested clinical isolates of typhoidal salmonellae, however the isolates suspected to be ESBL by phenotypic methods must be subjected further for molecular analysis. Key words: ESBL, typhoidal salmonellae, isolate.

Prevalence of multidrug‐resistant gram‐negative bacilli producing extended‐spectrum β‐lactamases (ESBLs) and ESBL genes in solid organ transplant recipients

Multidrug-resistant (MDR) gram-negative bacilli pose a serious and rapidly emerging threat to recipients of solid organ transplants (SOTs). However, extended-spectrum β-lactamases (ESBLs), as one of the cardinal mechanisms of resistance to antimicrobial agents in SOT recipients, remain obscure. The aim of this study was to investigate the prevalence of strains producing ESBLs in SOT patients with MDR gram-negative bacilli infections and to identify the ESBL genes carried by them. Isolates from various clinical specimens of SOT recipients were identified using the VITEK 2 system, and their antibiotic sensitivity was determined by the 2-fold agar dilution method. Isolates suspected of producing ESBL enzymes were tested by an ESBL phenotypic confirmatory method, and the ESBL genotypes were determined by the polymerase chain reaction and sequencing. A total of 80 MDR gram-negative strains were isolated from 350 SOT recipients. Among these strains, 42 (52.5%) had an ESBL-positive phenotype; 33 (42.3%) patients with ESBL-positive infection were found. Molecular analysis showed that most of the isolates harbored blaCTX-M-9 (78.6%), blaTEM (81.0%), and blaSHV (69.0%) genes. All the ESBL-producing strains were susceptible to carbapenems. MDR gram-negative isolates from SOT recipients are frequently ESBL producers. TEM and CTX-M9 were the predominant ESBL types.

VIRULENCE FACTORS AND DRUG RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM EXTRAINTESTINAL INFECTIONS

Purpose: To determine the virulence factors produced by Escherichia coli isolated from extraintestinal infections, to study the drug resistance pattern in E. coli with special reference to extended spectrum β-lactamase (ESBL) and to evaluate screening methods for ESBL. Methods: A total of 152 isolates of E. coli from various extraintestinal infections were screened for virulence factors such as haemolysin, surface hydrophobicity, serum resistance and protease. All the isolates were also studied for antibiotic susceptibility pattern using modified Kirby Bauer disk diffusion method. ESBL production was screened by standard disk diffusion method and confirmed using phenotypic confirmatory method. Results: Among 152 isolates, 36 (23.7%) were haemolytic, 42 (27.6%) were hydrophobic, 132 (86.8%) were serum resistant and only four were positive for protease. Multiple virulence factor were observed in 67 (44%) of isolates. Seventy-nine (51.4%) isolates produced ESBL. ESBL producing isolates showed multidrug resistance. There was a significant association (P < 0.001) between multiple virulence factors and ESBL production by extraintestinal E. coli. Conclusions: The present study shows the expression of virulence factors and multidrug resistance in E. coli isolated from various extraintestinal infections. The study also shows that appropriate methods of detecting drug resistance and ESBL production are required for the judicious use of antibiotics in managing these infections.

Evaluation of Four Commercially Available Extended-Spectrum Beta-Lactamase Phenotypic Confirmation Tests

Extended-spectrum beta-lactamase (ESBL) production in members of the Enterobacteriaceae can confer resistance to extended-spectrum cephalosporins, aztreonam, and penicillin. As such, the accurate detection of ESBL producers is essential for the appropriate selection of antibiotic therapy. Twenty previously characterized isolates and 49 clinical isolates suspected of ESBL production were tested by four ESBL phenotypic confirmatory methods for accuracy and ease of use. The four ESBL phenotypic confirmation tests included Dried MicroScan ESBL plus ESBL Confirmation panels (Dade Behring, Inc., West Sacramento, Calif.), Etest ESBL (AB BIODISK, Piscataway, N.J.), Vitek GNS-120 (bioMerieux, Inc., Hazelwood, Mo.), and BD BBL Sensi-Disk ESBL Confirmatory Test disks (BD Biosciences, Sparks, Md.). Results were compared to frozen microdilution panels prepared according to NCCLS specifications, and discrepant isolates were sent for molecular testing. The test sensitivities for the ESBL phenotypic confirmatory test methods used in this study were as follows: MicroScan ESBL plus ESBL confirmation panel, 100%; VITEK 1 GNS-120, 99%; Etest ESBL, 97%; and BD BBL Sensi-Disk ESBL Confirmatory Test disks, 96%. The test specificities were as follows: BD BBL Sensi-Disk ESBL Confirmatory Test disks, 100%; MicroScan ESBL plus ESBL confirmation panel and VITEK 1 GNS-120, 98%; and Etest ESBL, 94%. All methods were easy to perform; however, the Etest method required more expertise to interpret the results. All tests offer a feasible solution for confirming ESBL production in the clinical laboratory.

Screening and confirmatory testing for extended spectrum β-lactamases (ESBL) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca clinical isolates☆

Escherichia coli and Klebsiella spp. were screened for ESBL based on routine susceptibility testing results. Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporins or aztreonam were reported as probable ESBL producers. By using the NCCLS proposed ESBL confirmatory method, we tested 61 screen-positive isolates from 42 patients, 30 randomly selected susceptible isolates, and 12 isolates with previously characterized β-lactamases. Ceftazidime contributed to 97% of screen-positive isolates, whereas aztreonam added a single patient isolate. An ESBL was confirmed in 86% of K. pneumoniae, 100% of K. oxytoca, and 20% of E. coli screen-positive single patient isolates. None of the susceptible isolates were shown to produce ESBL. Based on these findings a comment regarding the presence of ESBL seems sufficient for Klebsiella spp. but confirmatory testing is indicated for E. coli. There was 85% agreement between the type of β-lactamase and the result of the ESBL confirmatory test. When a cefotaxime MIC > 0.25 μg/mL was used to indicate the presence of ESBL, the specificity of the assay increased to 100%. The NCCLS ESBL phenotypic confirmatory method was reproducible and accurate enough to be used in the clinical laboratory.