Prevalence of metallo-β-lactamase genes among Pseudomonas aeruginosa isolated from various clinical samples in China

AbstractBackgroundPseudomonas aeruginosa is an opportunistic pathogen which is associated with nosocomial infections and causes various diseases including urinary tract infection, pneumonia, soft-tissue infection and sepsis. The emergence of P. aeruginosa-acquired metallo-β-lactamase (MBL) is most worrisome and poses a serious threat during treatment and infection control. The objective of this study was to identify antibiotic susceptibility, phenotypic detection of MBL production and to determine the prevalence of MBL genes in carbapenem-resistant P. aeruginosa isolated from different clinical samples.MethodsA total of 329 non-duplicate P. aeruginosa isolated from various clinical samples from two hospitals in China between September 2017 and March 2019 were included in this study. Phenotypic detection of MBL was performed by the combined detection method using imipenem and imipenem-ethylenediaminetetraacetic acid (EDTA) discs. MBL-encoding genes including blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2, blaSPM-1, blaSIM, blaNDM-1 and blaGIM were detected by polymerase chain reaction (PCR).ResultsOf the 329 P. aeruginosa, majority of the isolates were resistant to imipenem (77.5%) followed by meropenem (64.7%). Of the 270 P. aeruginosa isolates tested, 149 (55.2%) isolates were found to be positive for MBL detection. Of the different samples, 57.8% (n = 26) of P. aeruginosa isolated from blood were found to be positive for MBL production. Of the various MBL genes, blaIMP-1 (28.2%) was the most predominant gene detected followed by blaVIM-2 (18.8%), blaVIM-1 (16.1%), blaNDM-1 (9.4%), blaIMP-2 (6.7%), blaSIM (6.0%), blaSPM-1 (4.0%) and blaGIM (1.3%) genes.ConclusionsThe high resistance of P. aeruginosa toward imipenem and meropenem and the high prevalence of blaIMP-1 and blaVIM-2 set the alarm on the increasing, perhaps the increased, carbapenem resistance. In addition to routine antibiotic susceptibility testings, our results emphasize the importance of both the phenotypic and genotypic MBL detection methods in routine practice for early detection of carbapenem resistance and to prevent further dissemination of this resistant pathogen.

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Investigation of Metallo-Beta-Lactamases in Carbapenem Resistant Pseudomonas aeruginosa Strains by Phenotypic and Genotypic Methods

Flora the Journal of Infectious Diseases and Clinical Microbiology ◽

10.5578/flora.68921 ◽

2020 ◽

Vol 25 (3) ◽

pp. 301-307

Author(s):

M. Duygu Aksoy ◽

H. Murat Tuğrul

Keyword(s):

Pseudomonas Aeruginosa ◽

Ethylenediaminetetraacetic Acid ◽

Carbapenem Resistance ◽

Beta Lactamase ◽

Bacterial Strains ◽

Beta Lactamases ◽

Carbapenem Resistant ◽

Synergy Test ◽

Phenotypic Tests ◽

Positive Controls

Introduction: Carbapenem resistant Pseudomonas aeruginosa strains cause serious problems in treatment. A large number of identified metallo-beta-lactamase (MBL) enzymes produced by P. aeruginosa are one of the most important mechanisms in resistance to carbapenems. MBL genes are located on the chromosome or plasmid, and they can easily spread between different bacterial strains. The activities of these enzymes are zinc-dependent, and they are inhibited by ethylenediaminetetraacetic acid (EDTA). Therefore, this advantage is used in MBL identification tests. In this study, it was aimed to determine MBL among P. aeruginosa strains. Materials and Methods: MBL existence was investigated in 35 P. aeruginosa strains accepted to be mildly susceptible/resistant to any of the carbapenem group of antibiotics through phenotypic and genotypic methods. Phenotypic tests were performed as double disk synergy test (DDST), combined disk diffusion tests (CDDT) by using 0.1 M and 0.5 M EDTA, MBL E-test, and modified Hodge test (MHT). blaIMP, blaVIM, blaGIM, blaSIM, blaSPM genes and blaNDM gene were investigated by multiplex polimerase chain reaction (PCR) and PCR, respectively. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 standard bacteria were used in tests. VIM-1, VIM-2, IMP-13, SPM-1, NDM-1 type MBL-producing P. aeruginosa strains were used as positive controls. Results: Among the carbapenems resistant P. aeruginosa isolates, positivity of MBL was found as 54.2% by MBL E-test, 42.8% by DDST, 94.2% and 37.1% by CDDT method using 0.5 M and 0.1 M EDTA, respectively. Modified Hodge test and genotypic method did not detect MBL. Conclusion: In order to correctly evaluate the results of the phenotypic method, the investigation of resistance genes by molecular methods is also required. The most common metallo-beta-lactamase enzymes responsible for resistance to carbapenem in Pseudomonas were not observed. It was thought that different mechanisms might be responsible for the identified carbapenem resistance.

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Molecular characterization of resistance mechanisms in Pseudomonas aeruginosa isolates resistant to carbapenems

The Journal of Infection in Developing Countries ◽

10.3855/jidc.9501 ◽

2018 ◽

Vol 11 (12) ◽

pp. 935-943 ◽

Cited By ~ 1

Author(s):

Mona Shaaban ◽

Ahmed Al-Qahtani ◽

Mohammed Al-Ahdal ◽

Rasha Barwa

Keyword(s):

Pseudomonas Aeruginosa ◽

Real Time ◽

Real Time Pcr ◽

Treatment Options ◽

Carbapenem Resistance ◽

Pulse Field ◽

Diffusion Method ◽

Clinical Samples ◽

Pcr Analysis ◽

Carbapenem Resistant

Introduction: Emergence of carbapenem resistance in Pseudomonas aeruginosa increases the therapeutic dilemma. In this study, we investigated various mechanisms involved in the resistance of P. aeruginosa clinical isolates to carbapenems. Methodology: P. aeruginosa isolates were isolated from different clinical samples. The antimicrobial susceptibility was evaluated by disc diffusion method. Carbapenemases were detected among carbapenem resistant isolates. Expression level of mexB and oprD was determined by real-time PCR. Molecular relatedness among isolates was detected based on pulse-field gel electrophoresis (PFGE). Results: Ninety P. aeruginosa isolates were purified from clinical specimens. High levels of resistance to imipenem and meropenem were detected in 16 isolates. PCR analysis of carbapenemases indicated the prevalence of Verona integron-encoded metallo-beta-lactamase (VIM); four isolates produced only VIM enzymes (VIM-1 or VIM-2), while the remaining twelve co-produced both VIM-1 or VIM-2 and NDM enzymes. Additionally, real-time PCR analysis elucidated high expression levels of mexB in seven of the carbapenem resistant isolates and low expression of oprD in seven isolates. The identified carbapenem-resistant isolates were clustered into eleven PFGE profiles where clusters E1 and E2 involved isolates exhibiting multiple carbapenemase genes (blaNDM-1, blaVIM-1 and blaVIM-2). Conclusion: Various mechanisms underlying carbapenem resistance have been detected in our P. aeruginosa cohort of isolates. Emergence of P. aeruginosa as a reservoir of multiple carbapenemases is increasing over time limiting the treatment options to this serious infection. This increases the urgency for infection control practices to reduce the incidence of this infection.

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Molecular Typing of Imipenem-ResistantAcinetobacter baumannii-calcoaceticusComplex in a Singapore Hospital Where Carbapenem Resistance Is Endemic

Infection Control and Hospital Epidemiology ◽

10.1086/518964 ◽

2007 ◽

Vol 28 (8) ◽

pp. 941-944 ◽

Cited By ~ 2

Author(s):

Thean Yen Tan ◽

Karen Poh ◽

Siew Yong Ng

Keyword(s):

Tertiary Care ◽

Carbapenem Resistance ◽

Diffusion Method ◽

Clinical Samples ◽

Care Hospital ◽

Typing Method ◽

Disk Diffusion Method ◽

Carbapenem Resistant ◽

High Prevalence ◽

Resistant Strains

Objective.To investigate the molecular epidemiology of carbapenem-resistantAcinetobacter baumannii-calcoaceticuscomplex isolates in a tertiary care hospital where the prevalence of carbapenem resistance among these organisms is high.Design.The study was a prospective, observational study performed during an 8-month period (May 1 through December 31, 2004).A. baumanniiisolates recovered from all clinical samples during the study period were included in the study. Antibiotic susceptibility testing was performed using the disk diffusion method, and all carbapenem-resistant strains were typed by a polymerase chain reaction-based typing method.Setting.An 800-bed hospital in Singapore.Results.More than half of recovered isolates were clonally unrelated, with the remaining isolates grouped into 4 genotypes.Conclusions.The results of the study suggest that the high prevalence of carbapenem resistance amongAcinetobacterorganisms in this institution is not caused by the spread of a predominant clone and that other factors may need to be investigated.

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Molecular Detection of bla OXA-48 Gene Encoding Carbapenem Resistance Pseudomonas aeruginosa Clinical Isolates from Khartoum State Hospitals, Sudan

10.1101/2020.06.22.20137034 ◽

2020 ◽

Author(s):

Doha Omer Ali ◽

Mohamed M.A. Nagla

Keyword(s):

Pseudomonas Aeruginosa ◽

Teaching Hospital ◽

Carbapenem Resistance ◽

Clinical Isolates ◽

Clinical Samples ◽

P Value ◽

Gene Encoding ◽

State Hospitals ◽

Carbapenem Resistant ◽

Different Strains

AbatractCarbapenem resistance in Pseudomonas.aeruginosa is particularly worrisome because this class of β-lactam represents the last therapeutic resource for control of bacterial infection.So this study aimed to detect the frequency of bla OXA-48 resistance gene among Pseudomonas aeruginosa clinical isolates during the period from November 2018 to November 2019.Hundred Pseudomonas aeruginosa clinical isolates, 81 carbapenems (imipenem meropenem) resistant and 19 carbapenems sensitive were collected from Omdurman Teaching Hospital, Fedail Hospital and Soba Teaching Hospital in Khartoum State-Sudan. All isolates were re-identified using conventional bacteriological techniques, their susceptibility to carbapenems were tested using Kirby-Bauer method for confirmation and investigated for the presence of the bla OXA-48 gene using conventional PCR technique.60 (60.0%) out of 100 Pseudomonas aeruginosa clinical isolates were positive for blaOXA-48 gene. Out of 81 carbapenem resistant isolates 54(66.7%) were positive for bla OXA-48 gene, while among the (19) carbapenem sensitive isolates 6 (31.6%) were positive for blaOXA-48 gene. There was statistically significant association between carbapenem resistant isolates and the presence of blaOXA-48 gene (P-value = 0.006).Wound swabs were the predominant clinical samples detected harboring bla OXA-48 gene both among the sensitive 5 (83.3%) and carbapenem resistant isolates 29(53.7) (P.value> 0.05).Our findings revealed high frequency of bla OXA-48 among carbapenem resistant isolates so identification of bla OXA-48 producing strains and taking efforts to reduce the rate of transferring these gene between the different strains is essential for optimization of therapy and improves of patients outcomes.

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Screening of Klebsiella Pneumoniae for Carbapenem Resistance and MIC of Imipenem

10.21203/rs.3.rs-36584/v1 ◽

2020 ◽

Author(s):

Sarada Saud ◽

Ashwani Agrawal ◽

Soniya Pokhrel ◽

Sushma Subedi ◽

Sanjit Shrestha ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Antibiotic Susceptibility ◽

Carbapenem Resistance ◽

Susceptibility Test ◽

Diffusion Method ◽

Clinical Samples ◽

Biochemical Tests ◽

Cross Sectional ◽

Carbapenem Resistant ◽

Antibiotic Susceptibility Test

Abstract Background: Klebsiella pneumoniae is a common opportunistic pathogen causing a wider range of infections, pneumonia, urinary tract infection, bacteremia, and liver abscesses; primarily in immunocompromised as well as immunocompetent individuals. This bacterium presents itself as an antibiotic resistant one especially in third generation cephalosporins and carbapenem, creating serious global challenges. Therefore, this cross-sectional study was conducted in B & B Hospital, Lalitpur with the aim to screening the distribution of carbapenem resistance Klebsiella pneumoniae through ertapenem and assess the minimum inhibitory concentration of imipenem for screened carbapenem positive K. pneumoniae. Methods: From 3447 different clinical samples collected according to standard guidelines, Klebsiella pneumoniae was identified through conventional microbiological techniques, staining and a panel of biochemical tests. The antibiotic susceptibility test of isolates was performed by the Kirby-Bauer disc diffusion method as per CLSI 2018 guidelines. Screening of carbapenem resistant was assessed by using ertapenem disc and the MIC of imipenem for carbapenem resistant and intermediate was done through epsilometer. Results: A total of 85 nonduplicate Klebsiella pneumoniae were identified and their antibiotic susceptibility test revealed that ceftriaxone was the least effective antibiotic. The number of MDR, carbapenem resistant and intermediate isolates was 51, 46 and 3, respectively. The MIC of imipenem through epsilometer from resistant and intermediate ertapenem isolates revealed that 31, 5 and 13 isolates were resistant, intermediate and sensitive, respectively.Conclusion: These findings showed the inconsistency in detection of carbapenem resistant isolates in routine microbiology laboratories and further support the other tests for detection of carbapenem resistance as suggested by CLSI.

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Comparative Study of Phenotypic Detection Methods of Metallo-beta-lactamase (MβL) Among Carbapenem Resistant Gram-Negative Bacilli (GNB) Isolated from Intensive Care Units (IUC) Patients

10.51429/ejmm29310 ◽

2020 ◽

Vol 29 (3) ◽

pp. 75-80

Author(s):

Raghda Hager ◽

Bassant M. Sayed

Keyword(s):

Intensive Care ◽

Intensive Care Units ◽

Carbapenem Resistance ◽

Detection Methods ◽

Diffusion Method ◽

Beta Lactamase ◽

Gram Negative ◽

Carbapenem Resistant ◽

High Prevalence ◽

Synergy Test

Background: Metallo-beta-lactamase (MβL) mediated resistance is an emergency threat in health care settings, and its identification is essential for treatment and infection control. Objectives: this study aimed to detect the MβL prevalence in Gram negative bacilli (GNB) and to compare its phenotypic detection methods. Methodology: Ninety six (96) isolates of GNB were isolated from different clinical specimens collected from patients admitted to intensive care units (ICU) of Ain Shams Hospitals, from February 2018 to June 2018. Isolates were screened for carbapenem resistance with imipenem 10 µg and meropenem 10 µg discs. The resistant isolates were tested for antibiotic susceptibility by disc diffusion method, and Meropenem minimum inhibitory concentration (MIC) were determinated, then the production of MβL was detected by imipenem-ethylene diamine tetra-acetic acid (EDTA) combined disc test (IPM-EDTA CDT), ceftazidime -EDTA combined disc test (CAZ-EDTA CDT) and Imipenem - EDTA double disc synergy test (IPM-EDTA DDST).Results: Forty three (43) isolates (44.7%) were resistant to carbapenem. Klebsiella pneumoniae (K.pneumoniae) was the most common isolated species; 29 (67.4%) isolates. Forty (40) isolates (93%) were positive for MβL by IPM-EDTA CDT method, whereas 36 (83.7%) were positive by CAZ -EDTA CDT method and 19 isolates (44.2%) were positive for MβL by IPM-EDTA DDST. Conclusion: High prevalence of MβL was detected among our isolates and IMP-EDTA CDT can be used as a phenotypic test in detection of MβL production.

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Phenotypic and Molecular Detection of IMP and SPM Metallo-Beta-Lactamases in Clinical Isolates of Carbapenem Resistant Pseudomonas aeruginosa

10.21203/rs.3.rs-60519/v1 ◽

2020 ◽

Author(s):

Zahra Norouzi Bazgir ◽

Mohammad Ahanjan ◽

Hamid Reza Goli ◽

Roya Ghasemian ◽

Mohammad Bagher Hashemi-Soteh

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Molecular Detection ◽

Clinical Isolates ◽

Beta Lactamase ◽

Antibiotic Susceptibility Pattern ◽

Beta Lactamases ◽

Phenotypic Identification ◽

Carbapenem Resistant ◽

High Prevalence

Abstract Objectives: Metallo-beta-lactamases play a major role in the resistance of Pseudomonas aeruginosa to carbapenems. The aim of this study was the phenotypic and molecular detection of IMP and SPM carbapenemase genes in 100 carbapenem-resistant clinical isolates of P. aeruginosa. The isolates identified using standard microbiological tests, and their antibiotic susceptibility pattern determined by disk agar diffusion (Kirby Bauer) method. Phenotypic identification of Metallo-beta-lactamase-producing strains assessed by the combined disk test (CDT). Then, PCR was used to detect the presence of IMP and SPM genes.Results: The highest and lowest levels of antibiotic resistance were observed against gentamicin (40%) and piperacillin-tazobactam (13%), respectively. Besides, 40 isolates (40%) had the Multi-drug Resistant (MDR) phenotype, while 5 (12.5%) MDR isolates were resistant to all antibiotics tested. The results of the CDT showed that among 43 carbapenem non-susceptible clinical isolates of P. aeruginosa, 33 (76.74%) isolates were Metallo-beta-lactamase-producing strains. Also, the frequency of the IMP gene was determined to be 9%, while none of these isolates carried the SPM gene. Due to the high prevalence of carbapenem-resistant and MDR P. aeruginosa in this study, routine antibiotic susceptibility testing and phenotypic identification of carbapenemase production by this bacterium are necessary for proper selection of antibiotics.

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Metallo - β - Lactamase Producers among Carbapenem Resistant Gram-Negative Isolates from Clinical Samples in a Tertiary Care Hospital

International Journal of Current Microbiology and Applied Sciences ◽

10.20546/ijcmas.2021.1009.012 ◽

2021 ◽

Vol 10 (9) ◽

pp. 105-113

Author(s):

M. Shabnum P. Sreenivasulu Reddy

Keyword(s):

Pseudomonas Aeruginosa ◽

Tertiary Care ◽

Carbapenem Resistance ◽

Control Measures ◽

Clinical Samples ◽

Care Hospital ◽

Klebsiella Species ◽

Strict Adherence ◽

Gram Negative ◽

Carbapenem Resistant

Gram – Negative Bacilli (GNB) are important cause of UTI, Blood stream infections, hospital acquired pneumonias. With the Carbapenems becoming the drug of choice in treating Multidrug resistant Organisms (MDRO) due to their safety and efficacy, there is rise in Carbapenem Resistant organisms which is becoming a threat to health care setup. Early diagnosis of Metallo – β – lactamase (MBL) producers by routine laboratory methods makes it the need of the hour to prevent spread of resistant strains. To detect MBL producers among Carbapenem resistant GNB. GNB were isolated from 2576 various clinical samples received by Department of Microbiology between December 2020 to March 2021. MBL production among Carbapenem resistant GNB was tested by Combined Disc Diffusion Assay using Imipenem disc and Imipenem + EDTA disc. Results: 899 GNB were isolated among 2576 samples with E. coli (35.05%) followed by Klebsiella species (28.58%) and Pseudomonas aeruginosa (14.90%). 180 isolates (20.02%) were Carbapenem Resistant GNB of which 55 isolates (30.55%) were MBL producers with Klebsiella species (29.01%) being highest MBL producer followed by Pseudomonas aeruginosa (27.27%). Rapid dissemination of MBL producers is worrisome making routine detection of MBL strains important. Regular surveillance, strict adherence to infection control measures and implementation of proper antibiotic policy is crucial to minimize the increasing Carbapenem resistance.

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Phenotypic Detection of Carbapenemase Production in Carbapenem-Resistant Enterobacteriaceae by Modified Hodge Test and Modified Strip Carba NP Test

Journal of Laboratory Physicians ◽

10.1055/s-0041-1723859 ◽

2021 ◽

Author(s):

Nisha Patidar ◽

Nitya Vyas ◽

Shanoo Sharma ◽

Babita Sharma

Keyword(s):

Carbapenem Resistance ◽

Nonparametric Test ◽

Multidrug Resistant ◽

Clinical Samples ◽

Chi Square ◽

Carbapenem Resistant ◽

The Social ◽

Significant Difference ◽

High Degree ◽

Carbapenem Resistant Enterobacteriaceae

Abstract Objective Carbapenems are last resort antibiotics for multidrug-resistant Enterobacteriaceae. However, resistance to carbapenem is increasing at an alarming rate worldwide leading to major therapeutic failures and increased mortality rate. Early and effective detection of carbapenemase producing carbapenem-resistant Enterobacteriaceae (CRE) is therefore key to control dissemination of carbapenem resistance in nosocomial as well as community-acquired infection. The aim of present study was to evaluate efficacy of Modified strip Carba NP (CNP) test against Modified Hodge test (MHT) for early detection of carbapenemase producing Enterobacteriaceae (CPE). Material and Methods Enterobacteriaceae isolated from various clinical samples were screened for carbapenem resistance. A total of 107 CRE were subjected to MHT and Modified strip CNP test for the detection of CPE. Statistical Analysis It was done on Statistical Package for the Social Sciences (SPSS) software, IBM India; version V26. Nonparametric test chi-square and Z-test were used to analyze the results within a 95% level of confidence. Results Out of 107 CRE, 94 (88%) were phenotypically confirmed as carbapenemase producer by Modified strip CNP test and 46 (43%) were confirmed by Modified Hodge Test (MHT). Thirty-eight (36%) isolates showed carbapenemase production by both MHT and CNP test, 56 isolates (52%) were CNP test positive but MHT negative, eight (7%) isolates were MHT positive but CNP test negative and five (5%) isolates were both MHT and CNP test negative. There is statistically significant difference in efficiency of Modified CNP test and MHT (p < 0.05). Conclusion Modified strip CNP test is simple and inexpensive test which is easy to perform and interpret and gives rapid results in less than 5 minutes. It has high degree of sensitivity and specificity. Modified strip CNP test shows significantly higher detection capacity for carbapenemase producers as compared with MHT.

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Complete Sequence of pOZ176, a 500-Kilobase IncP-2 Plasmid Encoding IMP-9-Mediated Carbapenem Resistance, from Outbreak Isolate Pseudomonas aeruginosa 96

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00423-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3775-3782 ◽

Cited By ~ 48

Author(s):

Jianhui Xiong ◽

David C. Alexander ◽

Jennifer H. Ma ◽

Maxime Déraspe ◽

Donald E. Low ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Carbapenem Resistance ◽

Complete Sequence ◽

Open Reading Frames ◽

Content Type ◽

Conserved Sequence ◽

Carbapenem Resistant ◽

Class 1 ◽

Usage Analysis ◽

Type 3

ABSTRACTPseudomonas aeruginosa96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Whole-genome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G+C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 ofPseudomonas fluorescensSBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. TheblaIMP-9-carrying integron containedaacA4→blaIMP-9→aacA4, flanked upstream by Tn21 tnpMRAand downstream by a completetnioperon of Tn402and amermodule, named Tn6016. The second integron carriedaacA4→catB8a→blaOXA-10and was flanked by Tn1403-liketnpRAand asul1-type 3′ conserved sequence (3′-CS), named Tn6217. Other features include three resistance genes similar to those of Tn5, a tellurite resistance operon, and twopiloperons. The replication and maintenance systems exhibit similarity to a genomic island ofRalstonia solanacearumGM1000. Codon usage analysis suggests the recent acquisition ofblaIMP-9. The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria.

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