Combined Disk Test Research Articles

Role of Multidrug-resistant Pathogens in Ventilator-Associated Pneumonia in a Tertiary Care Hospital in India

Aim: Ventilator-associated pneumonia (VAP) is the second most common infection acquired in the intensive care unit (ICU). Bacteriological profiles cause VAP and their susceptibility patterns vary in different institutions. Methods: A prospective study was conducted from June 2017 to May 2018 in a tertiary care hospital as per the recent NHSN guidelines in finding the incidence of VAP and further determining the etiological agents by both conventional and automated methods. The combination disk method (Phenotypic confirmatory test), ampicillin C (AmpC) disk test, modified carbapenem inactivation method, imipenem/ethylenediamine tetraacetic acid combined disc test, and cefoxitin disk test were performed for the detection of extended-spectrum beta-lactamases (ESBL), AmpC β-lactamases, carbapenemases, metallo-beta-lactamases (MBL), and methicillin-resistant Staphylococcus aureus, respectively. Results: Among 104 patients, 31 cases developed PVAP (possible VAP) during their ICU stay; of these cases, two patients had two episodes of VAP each, and the incidence of VAP was 32%. The most common isolate was Acinetobacter baumannii (38%), followed by Pseudomonas aeruginosa (22%), Klebsiella pneumoniae (16%), and Escherichia coli (13.51%). Twenty (54%) of the 37 VAP pathogens were multidrug resistant. ESBL was produced by 40% and 67% of E. coli and K. pneumoniae, respectively. MBL was produced by 25% of P. aeruginosa. In addition, AmpC beta-lactamases were produced by 18% each of the Enterobacteriaceae and non-fermenters, respectively. One of the two S. aureus isolates was methicillin-resistant. Conclusion: The majority of VAP cases in our setting were caused by highly resistant strains. The frequency of specific multidrug resistance pathogens causing VAP may vary due to hospital, patient population, exposure to antibiotics, type of ICU patients, and changes over time, emphasizing the need for timely local surveillance data.

The Status of Extended-spectrum β-lactamase and AmpC β-lactamase production in uropathogenic isolates of Escherichia coli and Klebsiella pneumonia in Bangladesh

Background: The rapid dissemination of antibiotic resistance as extended-spectrum beta-lactamases (ESBLs) and AmpC β-lactamases in bacteria became a major public health concern worldwide. Hence, this study was carried out to evaluate the status of uropathogenic isolates of Bangladesh especially Escherichia coli and Klebsiella pneumoniae in terms of ESBL and AmpC β-lactamases enzymes production. Methods: A laboratory-based cross-sectional survey was conducted with 553 urine samples at the Department of Microbiology, Popular Diagnostic Centre Ltd. Dhaka, Bangladesh for a period of 2.5 years from July 2017 to December 2019. Pathogens were obtained upon culture, and screened for ESBL production following the cephalosporin disk diffusion method and cephalosporin-clavulanate combination disc test (confirmatory) method. For AmpC β-lactamases enzymes, cefoxitin disc diffusion, and AmpC disc test (confirmatory) methods were performed. The positive isolates were further genotyped for the presence of blaTEM, blaSHV, blaOXA, and blaCTX-M β-lactamases genes in case of ESBL and blaFOX, blaCMY, and blaDHA genes for AmpC β-lactamases by polymerase chain reaction. Results: Of all urine samples, uropathogens were detected among 28.57% (158, n=553) samples. Among them, 46.20% (73) were E. coli and 20.25% (32) were K. pneumoniae. The prevalence of ESBL and AmpC β-lactamases production in E. coli was 34.24% (25) and 17.80% (13) respectively whereas it was 43.75% (14) and 21.87% (7) in Klebsiella pneumoniae isolates respectively. These resistance properties were further confirmed by phenotypic disc test. In case of ESBLs, most typical isolates possessed two or more resistance genes. Overall, the most prevalent gene was blaCTX-M (84.61%) followed by blaTEM (64.10%), blaOXA (46.15%) and blaSHV (25.64%). In case of AmpC β-lactamases FOX gene was most prevalent (45%) followed by CMY (20%) and DHA 3 (15%). Conclusion: The high prevalence of ESBL and AmpC β-lactamases production in our study emphasizes the judicious use of antibiotics in controlling urinary tract infections in Bangladesh.

Prevalence and Molecular Characterization of Metallo β-Lactamase Producing Gram-Negative Pathogens Causing Eye Infections.

Purpose:Metallo β-lactamases (MβL) production is a worldwide problem, particularly in gram-negative bacteria. As scanty data is available on the prevalence of MBL, the present study is being undertaken to determine the prevalence, antibacterial sensitivity patterns, and molecular characterization of MβL associated resistant genes in gram-negative bacteria isolated from ocular infections.Material and MethodsAt a tertiary eye care center in south India, 359 gram-negative pathogens, 200 isolates from eye infections, and 159 isolates from normal flora of the eye were studied. A gold standard microbiology method was used to identify the isolates. An antibiotic double disc synergy test and a combination disc test were used to detect MβL production. Multiplex PCR was used to investigate the molecular characteristics of the MβL encoding genes blaVIM, blaIMP, and blaNDM.ResultsOf the 359 gram-negative bacterial pathogens, Pseudomonas aeruginosa 108 (30.1%) and Enterobacter agglomerans 46 (12.8%) were commonly isolated. High prevalence of P. aeruginosa 81% (17 strains) was detected as an MβL producer and it shows 100% resistance to 2nd and 3rd generation cephalosporins and meropenem. Multiplex PCR detected only the blaVIM gene in 56 (28%) of various eye infections and 27 (17%) of normal flora of the gram-negative bacteria (GNB). The blaVIM gene is detected predominantly in 51.8% of keratitis and 21.4% of postoperative endophthalmitis. High prevalence of the gene was detected in P. aeruginosa 42.9% (24 of 56) and Alcaligens denitrificans 10.7% (6 of 56) from eye infections. Whereas, in the control group, P. aeruginosa and E. coli each had 14.8% (4 of 27) that were shown positive.ConclusionThe emerging MβLs mediated resistance among P. aeruginosa is a challenging task for ophthalmologists, especially in patients with endophthalmitis and bacterial keratitis. This local knowledge will aid in advising appropriate antibiotic use and avoiding unnecessary antibiotic prescriptions, which are highly warranted.

Detection of ESBLs types blaCTX-M, blaSHV and blaTEM resistance genes among clinical isolates of Pseudomonas aeruginosa

Pseudomonas (P.) aeruginosa is a leading cause of nosocomial infections in most part of the world. The production of extended spectrum β-lactamases (ESBLs) is an essential mechanism of ß-lactam resistance in these bacteria. This study aimed to investigate the prevalence of ESBL types blaTEM,blaSHV, and blaCTX-M in P. aeruginosa isolates from the patients hospitalized in Emam Reza Educational Hospital in Tabriz, northwest of Iran.A total of 100 P. aeruginosa nonduplicated isolates were collected from patients hospitalized in Emam Reza Educational Hospital. Antibiotic susceptibility was determined by the disk diffusion method. ESBL production was confirmed by ceftazidime discs alone and in combination with clavulanic acid (ceftazidime + clavulanic acid). The presence of ESBLs genes of blaTEM, blaSHV, and blaCTX-M were screened by Polymerase Chain Reaction (PCR) and sequencing tests. The DNA of bacteria was extracted and entered in a PCR reaction with primers specific for the proposed genes sequences.Among P. aeruginosa isolates, the highest antibiotic resistance was shown to ceftazidime 70 (70 %), and the highest susceptibility was shown to imipenem 55 (55 %). The results of combined disc test indicated that 66 % of isolates were ESBL producers. Screening for ESBLs genes revealed that ten strains (14 %) were positive for blaSHV, 26 % were positive for blaTEM, and 5 % isolates were positive for blaCTX-M genes.The results of this study showed a noteworthy prevalence of blaTEM,blaSHV, and blaCTX-M genes in the studied region that, suggests the necessity to take the preventing measures for controlling the dissemination of these resistant isolates.

A STUDY OF PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF NON FERMENTER GRAM NEGATIVE BACILLI ISOLATED FROM VARIOUS CLINICAL SAMPLES AT A TERTIARY CARE CENTER, JAIPUR

Introduction: Non-fermentative Gram-negative bacilli (NFGNB) are occasionally involved in infectious diseases pathology, but have shown resistance to multiple antibiotics and the capability to gain new resistance factors in the hospital environment. The present study was aimed to investigate the prevalence of Metallo-beta-lactamase (MBL) producing non-fermenters among the carbapenem-resistant isolates. AMethods: total of 200 NFGNB isolates from various clinical samples were analyzed by using different standard microbiological techniques and antimicrobial susceptibility using Kirby-Bauer method. MBL detection was performed by Modied Hodge test (MHT), imipenem-EDTA combined disc test, and modied Carbapenemase inactivation method (mCIM). In our study, 200 NFGNB isolates recovered from various clinical samples.Results: Pseudomonas aeruginosa was the most common NFGNB, accounting 112 (56%), followed by Acinetobacter baumannii 56 (28%), Burkholderia species 26 (13%) and other rare nonfermenter Gram negative bacilli 6 (3%). NFGNB isolates were maximally isolated from blood samples 73 (36.5%) followed by respiratory samples 46 (23%), urine 40 (20%), pus 37 (18.5%) and CSF 4 (2%). All the NFGNB isolates were susceptible to polymyxin B, colistin (100%), and cefoperazone + sulbactam (45.53%). Out of 112 Pseudomonas isolates, 77(68.75%) were imipenem resistant and of 56 Acinetobacter isolates, 35(62.5%) were imipenem resistant. Out of 77 imipenem resistant Pseudomonas 70 (90.9%) were MBL producers and out of 35 imipinem resistant Acinetobacter spp 32 (91.42%) were MBL producers. NFGNB are now emerging asConclusion: organisms of nosocomial infections. The use of broad-spectrum antibiotics should be avoided and quick detection and efcient infection control measures are essential to prevent the further spread of MBLs to other Gram-negative bacilli. Detection of MBL production and rationale antibiotic usage are the most important factors which control the gradually increasing NFGNB related infections

A Three-Year Look at the Phylogenetic Profile, Antimicrobial Resistance, and Associated Virulence Genes of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli is the most common cause of urinary tract infections, resulting in about 150 million reported annual cases. With multidrug resistance on the rise and the need for global and region surveillance, this investigation looks at the UPEC isolates collected for a 3-year period, with a view of ascertaining their antimicrobial susceptibility patterns and associated virulence determinants. The identification of bacteria isolates, antimicrobial susceptibility, and extended-spectrum beta-lactamases (ESBLs) production was determined with a Vitek 2 Compact Automated System (BioMerieux, Marcy L’Etoile, France). ESBLs were confirmed by the combined disc test (CDT) and basic biochemical test. The isolates were distributed into A (11%), B1 (6%), B2 (62.4%), and D (20.6%). Resistance to the penicillin group was high, between 88% and 100%. Additionally, resistance was high to cephalosporins (100%) in 2017 and 2018. The isolates were all sensitive to tigecycline, while resistance against imipenem and meropenem was low, at 4–12% in 2017 and 2018 and 0% in 2019. The results also showed that ESBL isolates were seen in 2017 and 2018. They were confirmed positive to CTX/CLA (88.5%) and CAZ/CLA (85%). By 2019, the number of resistant isolates reduced, showing only 4% ESBL isolates. Two virulence genes, fimH (46%) and papE/F (15%), were detected among the isolates by PCR. In conclusion, this study found that phylogroups B2 and D carried the most virulence genes as well as MDR and ESBL characteristics, suggesting the UPEC strains to be extraintestinal pathogens responsible for UTIs.

Detection of Carbapenem-resistant Enterobacteriaceae by Real-Time PCR and Assessment of Hospitalization Related Risk Factors Associated with their Fecal Carriage: A Case-control Study

Opportunistic behavior of commensal bacteria during severe infection, especially of the intestine is always considered as a predisposing threat for the severely ill patient admitted to hospitals. A descriptive-analytical case-control study was carried out to detect the prevalence and risk factor analysis of fecal carriage of Carbapenem-resistant Enterobacteriaceae. Patients having fecal carriage of Carbapenem-resistant Enterobacteriaceae were considered as cases and patients who were without Carbapenem-resistant Enterobacteriaceae were taken as the control in a proportion of 1:4. Carbapenem-resistant Enterobacteriaceae (CRE) was confirmed by both conventional as well as molecular methods. Methods such as Combined Disk Test, mCIMtest, and RAPIDEC CarbaNP Test were used for phenotypic identification of CRE, Whereas Real-Time (RT)-PCR was used for the detection of bla-gene encoded for CRE (blaNDM1, blaVIM, blaKPC, blaIMP, and blaOXA48). All patients belonging to medicine wards were included in the study. we screened 436 patients during the study and found 160 patients suitable for our study, out of which 32 (20%) were cases and 128 (80%) were controls. We found a total number of 25 genes out of 16 isolates, where NDM 1 was identified in maximum numbers followed by KPC &VIM. Standard statistical analyses such as chi-squire and odd ratios were conducted to determine the risk factor of different variables. Stepwise multiple logistic regressions were carried out, where we found, that transfer from other wards to medicine ward, use of nebulizer and intravenous catheter, and use of multiple antibiotics were still statistically significant. Implementation of Multi-modal colonization prevention and control is the need for the present situation throughout the world.

Dissemination and Genetic Relatedness of Multidrug-Resistant and Extensively Drug-Resistant Acinetobacter baumannii Isolates from a Burn Hospital in Iraq.

Acinetobacter baumannii is an aggressive opportunistic bacterial pathogen that causes severe nosocomial infections, especially among burn patients. An increasing number of hospitals-acquired infections have been reported all over the world. However, little attention has been paid to the relatedness between A. baumannii isolates from different hospital environments and patients. In this study, 27 isolates were collected from the Burn and Plastic Surgery Hospital of Al Sulaymaniyah City, Iraq, from January through December 2019 (11 from patients and 16 from the wards environment), identified to species level as A. baumannii using Vitek 2 system and molecular detection of 16S rRNA gene, and then confirmed by targeting the blaOXA-51 gene. Moreover, the isolates were characterized by means of automated antimicrobial susceptibility assay, antimicrobial-resistant patterns, a phenotypic method using a combined disk test, and molecular methods for the detection of class A and C β-lactamase genes, and finally, the genetic relatedness was classified. Antimicrobial susceptibility testing showed that 63% (17/27) of the retrieved A. baumannii isolates were extensively drug-resistant to 8/9 antimicrobial classes. Furthermore, 37% (10/27) of the isolates were classified as multidrug-resistant; 8 isolates exhibited similar resistant patterns and the other two isolates showed 2 different patterns, while resistance was greater in isolates from patients than from the ward environment. Combined disk test showed that two isolates contained extended-spectrum β-lactamase. All isolates carried blaTEM-1, and two copies of the blaCTX-1 gene were indicated in one isolate, while blaSHV was absent in all isolates. Twenty-four isolates carried the blaAmpC gene; among them, 3 isolates harbored the insertion sequence ISAba-1 upstream to the gene. Using Enterobacterial Repetitive Intergenic Consensus PCR, the isolates were clustered into 6 distinct types; among them, two clusters, each of four strains, were classified to contain isolates from both patients and environments. The clusters of similar genotypes were found in inpatients as well as the environments of different wards during time periods, suggesting transmission within the hospital. Identification of possible infection sources and controlling the transmission of these aggressive resistance strains should be strictly conducted.

Detection of carbapenemases blaOXA48-blaKPC-blaNDM-blaVIM and extended-spectrum-β-lactamase blaOXA1-blaSHV-blaTEM genes in Gram-negative bacterial isolates from ICU burns patients

Background and objectivesBurn patients are highly susceptible to invasion by multidrug-resistant Gram-negative bacteria (MDR-GNB) through post-burn damage. The prevalence of MDR-GNB isolated from burns patients has increased dramatically in the last decade, representing a serious risk to patients admitted to burns units worldwide. The challenges of managing infected burns patients are exacerbated in poor resource settings. This study was designed to develop a pathway for the rapid diagnosis of multidrug-resistant (MDR) Gram-negative infections and identify the bacterial genes including blaOXA1, blaTEM, and blaSHV encoding ESBLs and blaOXA48, blaKPC, blaNDM, and blaVIM encoding carbapenemases from the patient of post burns infection. MethodsClinical isolates were collected (August 2017 to August 2018) from Intensive care unit (ICU) of Burn Centre. Antibiotic susceptibility testing and phenotypic detection of ESBLs and carbapenemases was performed by disk diffusion, double disk synergy test (DDST), combination disk test (CDT), and Imipenem + EDTA combined disk test (IMP + EDTA CDT). Polymerase chain reaction (PCR) detection was performed for ESBLs blaOXA1-blaSHV-blaTEM and carbapenemases genes blaOXA48-blaKPC-blaNDM-blaVIMResultsIn total, of 170 Gram-negative isolates, 104 (61.2%) were confirmed as multidrug-resistant (MDR); Pseudomonas aeruginosa was found to be the most prevalent 43/104 (41.4%), followed by Klebsiella pneumoniae 17/104 (16.4%), Acinetobacter baumannii12/104 (11.5%), and 6/104 Proteus mirabilis (5.8%). All isolates (100%) were resistant to cefotaxime and ceftazidime, while the meropenem resistance was 58.7%. ESBL and carbapenemase genotypes were found to be associated with higher MAR index (0.65–0.88) and MIC (> 32 µg/ml) values P. aeruginosa was the major ESBL and carbapenemase producer as determined by phenotypic testing and PCR. blaTEM positive isolates among ESBLs producers were predominant 81.8% (27/33), followed by 27.3% blaOXA1 and blaSHV, respectively. blaVIM positive isolates among carbapenemase producers were predominant 47.7% (21/44), followed by 27.3% blaKPC, 20.5% blaOXA48, and 11.4% blaNDM positive isolates.ConclusionsThe predominant organism causing burn infections was ESBL and carbapenemase-producing Pseudomonas aeruginosa. There are only limited effective antibiotics against such strains. blaVIM and blaTEM individually and in co-existence with blaKPC, blaOXA48, blaSHV, and blaOXA1 confer antimicrobial resistance in burns patients. Rapid detection of ESBL and carbapenemase genes will inform treatment strategies improving the outcome for post-burn patients in ICU.

Biofilm Production and its Association with Multi Drug Resistance in Pseudomonas aeruginosa among ICU Patients with Special Reference to ESBL, AmpC and MBL production

Multidrug resistant Pseudomonas aeruginosa is an alarming and emerging public health problem globally across the developing countries. Pseudomonas aeruginosa is still a major cause for nosocomial infection and approx 10-20% of these patients are admitted to the ICU’s. Bacterial isolates those are biofilm producers are more drug resistant than biofilm Non-producers. The aim of the present study was to evaluate the production of biofilm and β-lactamases (ESBL, MBL, AmpC) in multi drug resistant Pseudomonas aeruginosa isolated from ICU patients. The present cross-sectional prospective study was carried out in the Department of Microbiology, Santosh Medical College & Hospital, Ghaziabad, Uttar-Pradesh, India. A total of 115 isolates of P. aeruginosa were isolated from 502 clinical samples. After confirmation of MDR status of P. aeruginosa further processing for biofilm and beta lactamases was performed accordingly. Biofilm production was done by test tube method and tissue culture plate method along with phenotypic profiling of ESBL, MBL and AmpC was performed by disc potentiation test; IMP-EDTA combined disc test and Cefoxitin Cloxacillin Double Disc synergy test (CC-DDST) respectively. Out of 502 total human clinical samples 115 isolates were Pseudomonas aeruginosa giving the prevalence rate of 23%. Among 115 isolates of P. aeruginosa 60 (52%) were MDR phenotypes, Out of 60 MDR isolates 23 (38.3%) were ESBL producers, 22 (36.6%) were MBL producers, and 3(5%) were AmpC producers. Out of total 115 isolates 68(59%) isolates were biofilm producers and 47 (40.8%) were biofilm non-producers. Strict antibiotic policies with early detection of beta lactamases and detection of biofilm production should be performed regularly for all clinical isolates of Pseudomonas aeruginosa so as to guide antibiotic selection along with better management of severe infection in ICU patients.

Phenotypic detection of metallo-beta-lactamase production in clinical isolates of Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital

Introduction: Carbapenem resistance due to metallo-beta-lactamase (MBL)-producing bacteria is an emerging threat worldwide. This study aimed to detect the MBL production in clinical isolates of E. coli and Klebsiella pneumoniae species in our hospital setting and to evaluate the efficiency of two phenotypic methods for the detection of MBL production. Materials and Methods: The present study was carried out in the Department of Microbiology, MGM Medical College and Hospital, Aurangabad, Maharashtra, for a period of 2 years from April 2018 to March 2020. From a total of 12,324 various clinical specimens, 393 isolates of E. coli and Klebsiella pneumoniae species were tested for MBL production. MBL was detected by two different phenotypic methods, i.e., combined disc test and E-test. Results: Out of 393 isolates, 130 (33.07%) isolates were resistant to imipenem on screening of which 71 (18.06%) were Klebsiella pneumoniae and 59 (15.01%) were E. coli. About 43.66% Klebsiella pneumoniae isolates and 40.67% E. coli isolates were MBL-positive by the combined disc test. Using the E-test, MBL production was found to be 46.47% and 45.76% in Klebsiella pneumoniae and E. coli, respectively. Conclusion: Routine screening of MBL-producing organisms should be performed in diagnostic laboratories to control the spread of resistance and for the proper management of antibiotic therapy. E-test is better than the combined disc test for the detection of MBL-producing gram-negative bacilli.

Novel insights into pivotal risk factors for rectal carriage of extended-spectrum-β-lactamase-producing enterobacterales within the general population in Lower Saxony, Germany.

To estimate the prevalence of extended-spectrum-β-lactamase (ESBL)-producing enterobacterales (ESBL-E) carriage in the general population of Lower Saxony, Germany, and to identify risk factors for being colonized. Participants were recruited through local press and information events. Detection of ESBL-E by culture was conducted using ESBL-selective chromagar plates containing third-generation cephalosporins. Identification of pathogens was performed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)_technology on Vitek mass spectrometry. Antibiotic susceptibility testing was conducted by microdilution (Vitek II) and an ESBL confirmation assay was carried out using a combination disk test. Of 527 randomly collected stool samples from healthy volunteers, 5.5% were tested positive for ESBL-E. Post-stratification for age and gender yielded a similar population estimate (5.9%). People traveling abroad and taking antibiotics had the greatest rectal ESBL-E carriage. Potential risk factors (eg, working in healthcare facilities, recent inpatient stay) did not attribute to rectal ESBL-E carriage as other factors (eg, travelling, taking antibiotics). Rectal ESBL-E carriage within the general population seems to be high. The known risk factors for carriage with MDRO might not be fully applicable to ESBL-E and require further examination in order to develop effective strategies for the prevention of ESBL-E dissemination within the general population.

The prevalence of plasmid-mediated quinolone resistance genes among Escherichia coli strains isolated from urinary tract infections in southwest Iran.

The extensive and inappropriate use of quinolones, which are frequently used as an effective treatment for urinary tract infection (UTI) patients, has led to resistance to these antibiotics. This study was designed to determine the prevalence of quinolones resistance and the presence of plasmid-mediated quinolone resistance (PMQR) genes among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates. One hundred and fourteen E. coli isolates were collected from patients' urine samples. The susceptibility of isolates to selected antibiotics was tested by the Kirby-Bauer disk diffusion method. ESBL-producing isolates were identified phenotypically using a combination disk test. Using specific primers, the frequency of aac (6')-Ib, qnrA, qnrB, qnrC, qnrD, qnrS, and qepA genes was investigated by polymerase chain reaction (PCR). Among 26 ESBL-producing isolates, the highest resistance rate was observed toward nalidixic acid (80.8%) and ciprofloxacin (61.5%), respectively. Ninety-seven (85%) of all isolates harbored at least one PMQR gene, the most frequent one being aac(6')-Ib-cr variant (47.4%). Coexistence of aac(6')-Ib-cr variant and qnrB were the most broadly distributed genotype among quinolone resistance isolates. Notably, none of the isolates contained the qnrC, qnrD, and qepA genes. Our results highlight the significant prevalence of PMQR genes in ESBL-producing E. coli isolates in this region. Also, the aac (6')-Ib-cr variant was the most frequent gene, particularly in ESBL positive isolates. A regular periodic monitoring program is needed to control and hinder the more spread of antibiotic resistance phenomenon and contributed genes among UTI-causing E. coli isolates.

Comparison of the Performance of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) in Clinical Practice

In order to investigate the diagnostic performance characteristics of four phenotypic assays in detecting carbapenem-resistant Enterobacteriaceae (CRE), we collected the CRE strains from infected patients. The results of carbapenemase gene detection, bla KPC-2, bla OXA-23, bla NDM-1, bla NDM-4, bla NDM-5, bla IMP-4, and bla IMP-8, were used as a standard to evaluate the performances of combined disk test (CDT), modified carbapenem inactivation method(mCIM)/EDTA-modified carbapenem inactivation method(eCIM), NG-Test CARBA 5 (CARBA), and color developing immunoassay (CDI). The compliance of phenotype results based on CDT, mCIM/eCIM, CARBA, and CDI with genetic detection results was 94% (231/247), 95% (235/247), 98% (242/247), and 99% (246/247), respectively. CDT demonstrated a low specificity for carbapenemase detection, low negative predictive value (NPV), and low sensitivity for metallo-β-lactamase (79%, 55%, and 88%, respectively); it also failed to accurately detect IMP. The mCIM/eCIM assay had serious problems in detecting OXA-23-like carbapenemases. The sensitivity and specificity of CARBA and CDI were higher than those of the first two methods. However, CARBA did not cover the detection of OXA-23, while CDI cannot detect IMP-8, resulting in low NPVs (70% and 88%, respectively). In conclusion, CARBA and CDI assays are highly accurate except individual rare genes and allow direct genotype detections. CDT and mCIM/eCIM assays are moderately accurate and can only distinguish serine-β-lactamases from metallo-β-lactamases. Laboratories should choose the appropriate method that meets their needs based on its characteristic.

Phenotypic characterization of Acinetobacter species and their resistance pattern in a tertiary care hospital

is an emerging opportunistic pathogen and increasingly implicated in hospital acquired infection especially in intensive care unit. The infection caused by spp. is difficult to control due to multi drug resistance which limits its therapeutic options. This study was undertaken to isolate from various clinical samples and to speciate isolates. Efforts were made to study the antimicrobial susceptibility pattern of species and to detect metallo beta lactamase producing strains. Clinical samples blood, urine, sputum, pus, body fluids and ETT sample received in microbiology laboratory were processed according to the standard laboratory procedure. Identification and antimicrobial susceptibility testing was done by VITEK-2 automated method. All the isolates that showed resistance to Imipenem were tested for Metallo – Beta – Lactamase (MBL) production by phenotypic test, Imipenem-EDTA-combined disc test method. Out of total 100 spp isolated from clinical samples, 80 were isolated from critical care units. baumannii was common spp 88 (88%), followed by A.complex 10 (10%). Isolation rate of species was highest from blood (27%) specimens followed by ETT (19%). 44% strains of were isolated from respiratory samples. 84(84%) spp, were carbapenem resistant. Out of 84 carbapenems resistant strains 48 (57%) were MBL positive strains by combined disc test. Emergence of MBLs producing strains of is alarming and reflects excessive use of carbapenems. Therefore early detection and prompt infection control measures is important to prevent further spread of MBLs to other Gram negative bacilli.

Diffusion of OXA-48 carbapenemase among urinary isolates of Klebsiella pneumoniae in non-hospitalized elderly patients

BackgroundRecently, a dramatic increase of Klebsiella pneumoniae positive for OXA-48 β-lactamases was observed first in the hospital setting and later in the long-term care facilities (LTCFs) and community in the Zagreb County, particularly, in urinary isolates. The aim of the study was to analyse the epidemiology and the mechanisms of antibiotic resistance of OXA-48 carbapenemase producing K. pneumoniae strains isolated from urine of non-hospitalized elderly patients.ResultsThe isolates were classified into two groups: one originated from the LTCFs and the other from the community. Extended-spectrum β-lactamases (ESBLs) were detected by double disk-synergy (DDST) and combined disk tests in 55% of the isolates (51/92). The ESBL-positive isolates exhibited resistance to expanded-spectrum cephalosporins (ESC) and in majority of cases to gentamicin. LTCFs isolates showed a significantly lower rate of additional ESBLs and consequential resistance to ESC and a lower gentamicin resistance rate compared to the community isolates, similarly to hospital isolates in Zagreb, pointing out to the possible transmission from hospitals.ESBL production was associated with group 1 of CTX-M or SHV-12 β-lactamases. Ertapenem resistance was transferable from only 12 isolates. blaOXA-48 genes were carried by IncL plasmid in 42 isolates. In addition IncFII and IncFIB were identified in 18 and 2 isolates, respectively. Two new sequence types were reported: ST4870 and ST4781.ConclusionsThis study showed eruptive and extensive diffusion of OXA-48 carbapenemase to LTCFs and community population in Zagreb County, particularly affecting patients with UTIs and urinary catheters. On the basis of susceptibility testing, β-lactamase production, conjugation experiments, MLST and plasmid characterization it can be concluded that there was horizontal gene transfer between unrelated isolates, responsible for epidemic spread of OXA-48 carbapenemase in the LTCFs and the community The rapid spread of OXA-48 producing K. pneumoniae points out to the shortcomings in the infection control measures.

Genomic Analysis of Highly Virulent blaCTX-M, blaSHV and blaTEM Genes in Resistant Strains of E.coli and Klebsiella: an emerging threat

Background: The term antimicrobial resistance refers to the ability to resists the effects of drugs formally used to treat them and this term relates only to bacteria becoming resistant. Microorganisms that are resistant to multiple drugs are known as multidrug-resistant bacteria. Aim: To investigate the prevalence of SHV, TEM and CTXm genes from E.coli and Klebsiella isolated from patients of Lahore, Pakistan. Methods: Patients with prolonged hospital stay were enrolled in this cross-sectional study with 60 samples comprising Klebsiella pneumoniae and Escherichia coli (Ctx, Cro, Caz resistant) were identified in clinical specimens. To assess susceptibility, the disc diffusion method was applied with eight antibiotic panel of cephalosporin 3rd generation. A Double disc, combined disc test was used to identify the ESBL-producing bacteria. By real - time PCR, The presence of the genes encoding blaTEM, blaSHV, and blaCTXm was tested in ESBL positive isolates as well as additional isolates with MICs of less than 4g/mL for ceftazidime, cefotaxime, ceftriaxone, and aztreonam (PCR). Results: The frequency of E. coli and Klebsiella bacteria was found in 59% and 41% of the 60 samples, respectively. According to the data, 23 isolates (16.37%) were multidrug resistant, and 7(6.89%) were ESBL-positive. At least one of the antibiotics ceftazidime, ceftriaxone, or cefotaxime was resistant to 30(25.86%) of the isolates. The ESBL genes were sequenced to corroborate the PCR result. Conclusions: Among E. coli and Klebsiella bacteria obtained from patients, blaTEM-116 was the most frequently isolated ESBL gene, followed by Shv and Ctxm. Keywords: Antibiotic Resistance, Beta- Lactamases, E. coli, Klebsiella pneumonia

A comparative study of VITEK-2, Double Disc Synergy and Combined Disc Methods for detection of ESBL (Extended Spectrum Beta-Lactamase) production in Escherichia coli and Klebsiella pneumoniae strains

Aim: In this prospective study we aimed to compare the effectiveness of VITEK-2 (bioMérieux, France) automated system, double-disc synergy (DDST) versus combined disc test (CDT) in detecting the Extended-Spectrum Beta-Lactamase (ESBL) positivity in Escherichia coli and Klebsiella pneumoniae strains isolated from various clinical samples. Material and Method: E.coli and K. pneumoniae strains inoculated on Mueller Hinton Agar plate. Susceptibility tests were performed with the VITEK 2 (BioMérieux, France) system before. Afterward, EBSL positivity was investigated manually DDST and CDT. Minimal inhibitor concentration (MIC) results of three tests were compared with each other according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Results: 184 E. coli and K.pneumoniae strains, elevated. 92.9% of 98 patients with VITEK 2 positive results were positive with combined disc and DDS method, 100% of the 86 patients with negative results of VITEK 2 were negative with combined disc and MDC. Conclusion: VITEK 2 was found to have a sensitivity of 100%, a specificity of 92.4%, a positive predictive value of 92.8% and a negative predictive value of 100%. VITEK 2 was found to be compatible with validation tests for ESBL positivity.

Detection of Colistin Resistance in Uropathogenic Carbapenem Resistant Klebsiella pneumoniae

Background: Colistin resistance among carbapenem-resistant Klebsiella pneumoniae (CRKp) strains at an accelerated rate poses a serious global threat which limits therapeutic options. Objective: In this study, 203 laboratory-archived uropathogenic K. pneumoniae isolates were screened for carbapenem and colistin resistance. Methods: Carbapenam resistance screening was performed using combined disc test. Colistin resistance was determined for CRKp isolates, according to clinical and laboratory standards institute- European Committee on Antimicrobial Susceptibility Testing guidelines. Polymerase Chain Reaction for carbapenamase genes (blaNDM-1, blaKPC-2 and blaOXA-1) and plasmid-mediated colistin resistance genes (mcr-1-2) were performed for the resistant isolates. Results: About 30% of the CRKp isolates were resistant to colistin; 57.1% and 35.7% of the isolates carried blaNDM-1 and blaOXA-1 gene, respectively. None of the isolates showed positive for blaKPC-2 and mcr (1-2) genes. Conclusion: Colistin resistance in the absence of a plasmid borne transfer mechanism was observed among our CRKp strains. Escalating colistin resistance is a crucial obstacle in treatment of K. pneumoniae infections and repurposing their usage is important to combat and control the resistance.

Predominance of Acinetobacter spp., Harboring the blaIMP Gene, Contaminating the Hospital Environment in a Tertiary Hospital in Mwanza, Tanzania: A Cross-Sectional Laboratory-Based Study.

Data on colonization and hospital contamination of carbapenem-resistant Gram-negative bacteria (CR-GNB) are limited in low- and middle-income countries. We designed this study to determine the prevalence and co-existence of carbapenemase genes among CR-GNB isolated from clinical, colonization, and hospital environmental samples at a tertiary hospital in Mwanza, Tanzania. The modified Hodge test (MHT), the combined disk test (CDT), and the double-disk synergy test (DDST) were used for the phenotypic detection of carbapenemases. A multiplex PCR assay was used to detect blaIMP and blaKPC, and a singleplex PCR assay was used to detect blaOXA-48. Data were analyzed by STATA version 13.0. Overall, 68.8% (44/64) of the CR-GNB had at least one phenotype by phenotypic methods, whereby 60.9% (39/64) were both CDT and DDST positive and 31.3% (20/64) were MHT positive. A total of 23/64 (35.9%) had at least one of the genes tested with the predominance of blaIMP (91.3%; 21/23). In addition, 47.7% (21/44) of the CR-GNB phenotypes had at least one gene. Around 47.8% (11/23) of the CR-GNB carried multiple genes encoding for carbapenem resistance, with the maximum co-existence of blaIMP/blaKPC/blaOXA-48 (45.5%; 5/11). The majority of carbapenem-resistant genes were detected in Acinetobacter spp. (82.6%; 19/23) and isolated from bed swabs (69.6%; 16/23). Acinetobacter spp. carrying the blaIMP gene predominantly contaminated the hospital environment. Therefore, we recommend routine decontamination of inanimate hospital surfaces, including patient beds.