Presence Of Carbapenemase Genes Research Articles

Molecular characteristics of carbapenem-resistant Enterobacteriaceae isolated from two tertiary hospitals in the Philippines

Species belonging to the family Enterobacteriaceae cause infections that result in clinically significant morbidity and mortality. These organisms have been shown to harbor multiple mechanisms of resistance to antibiotics, including carbapenems. Molecular characterization of resistance to carbapenems is critical since few effective therapeutic options exist, most of which are difficult to access in resource-limited settings. One hundred twenty-six Enterobacteriaceae isolates with phenotypic resistance to at least one carbapenem were collected from two tertiary hospitals in the Philippines from 2014 to 2016. The organisms were screened for the presence of genes coding for carbapenemases and extended spectrum β-lactamases (ESBLs) using multiplex PCR. Multilocus sequence typing (MLST) was performed for Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae complex. The majority of the carbapenem-resistant isolates were K. pneumoniae (70/126, 55.5%) followed by K. ozaenae (19/126, 15%). Amplification of genes conferring carbapenem resistance showed blaNDM in 89 isolates (70%), followed by blaKPC in 21 isolates (16%). MLST revealed the presence of different clones including ST16 (5/70), ST17 (3/70) and ST147 (9/70) in K. pneumoniae, and ST10 (3/13) in E. coli. This study shows that blaNDM is the most prevalent carbapenemase type among carbapenem-resistant Enterobacteriaceae (CRE). Presence of carbapenemase gene and multiple ESBLs in the same organism pose a risk in the clinical setting since it could confer higher antibiotic resistance. This high rate of resistance poses the risk of spread since it can be transferred by mobile genetic elements. This study illustrates the acute need for effective therapeutic and hospital infection controls against CREs.

Predictive Modeling of Phenotypic Antimicrobial Susceptibility of Selected Beta-Lactam Antimicrobials from Beta-Lactamase Resistance Genes.

The outcome of bacterial infection management relies on prompt diagnosis and effective treatment, but conventional antimicrobial susceptibility testing can be slow and labor-intensive. Therefore, this study aims to predict phenotypic antimicrobial susceptibility of selected beta-lactam antimicrobials in the bacteria of the family Enterobacteriaceae from different beta-lactamase resistance genotypes. Using human datasets extracted from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program conducted by Pfizer and retail meat datasets from the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), we used a robust or weighted least square multivariable linear regression modeling framework to explore the relationship between antimicrobial susceptibility data of beta-lactam antimicrobials and different types of beta-lactamase resistance genes. In humans, in the presence of the blaCTX-M-1, blaCTX-M-2, blaCTX-M-8/25, and blaCTX-M-9 groups, MICs of cephalosporins significantly increased by values between 0.34-3.07 μg/mL, however, the MICs of carbapenem significantly decreased by values between 0.81-0.87 μg/mL. In the presence of carbapenemase genes (blaKPC, blaNDM, blaIMP, and blaVIM), the MICs of cephalosporin antimicrobials significantly increased by values between 1.06-5.77 μg/mL, while the MICs of carbapenem antimicrobials significantly increased by values between 5.39-67.38 μg/mL. In retail meat, MIC of ceftriaxone increased significantly in the presence of blaCMY-2, blaCTX-M-1, blaCTX-M-55, blaCTX-M-65, and blaSHV-2 by 55.16 μg/mL, 222.70 μg/mL, 250.81 μg/mL, 204.89 μg/mL, and 31.51 μg/mL respectively. MIC of cefoxitin increased significantly in the presence of blaCTX-M-65 and blaTEM-1 by 1.57 μg/mL and 1.04 μg/mL respectively. In the presence of blaCMY-2, MIC of cefoxitin increased by an average of 8.66 μg/mL over 17 years. Compared to E. coli isolates, MIC of cefoxitin in Salmonella enterica isolates decreased significantly by 0.67 μg/mL. On the other hand, MIC of ceftiofur increased in the presence of blaCTX-M-1, blaCTX-M-65, blaSHV-2, and blaTEM-1 by 8.82 μg/mL, 9.11 μg/mL, 8.18 μg/mL, and 1.04 μg/mL respectively. In the presence of blaCMY-2, MIC of ceftiofur increased by an average of 10.20 μg/mL over 14 years. The ability to predict antimicrobial susceptibility of beta-lactam antimicrobials directly from beta-lactamase resistance genes may help reduce the reliance on routine phenotypic testing with higher turnaround times in diagnostic, therapeutic, and surveillance of antimicrobial-resistant bacteria of the family Enterobacteriaceae.

Association of Diabetes Mellitus With Increased Mortality in Carbapenem-Resistant Enterobacterales Infections.

Introduction Carbapenem-resistant Enterobacterales (CRE) infections have high mortality. We aimed to examine the diabetes mellitus (DM) association with CRE mortality. Methodology Our study is a retrospective cohort study including patients who were admitted to the medical wards in the main district hospital (New Jahra Hospital, Kuwait) between January 1, 2022, and January 1, 2023, and diagnosed with CRE infections during hospitalization. The patients were divided into diabetic and non-diabetic groups. Clinical and laboratory data were collected. The presence of carbapenemase genes was detected. The primary outcome was 30-day hospital mortality. We assessed the effect of glycemic control on the outcomes. Results We included 47 patients in the diabetic group and 39 patients in the non-diabetic group. Females represented 54.7% of patients, and the median age was 73 and 55 years in the two groups, respectively. Klebsiella pneumonia (86%) and Escherichia coli (12.8%) were the most frequently isolated CRE. Carbapenemase genes were detected in all patients: NDM-1 in 67.4%, OXA-48 in 18.6%, and both genes coexisted in 14%. The 30-day hospital mortality was significantly higher in the diabetic group compared to the non-diabetic group (48.9% vs. 28.2%, P = 0.041). Among the diabetic patients, there was no significant difference between survivors and non-survivors regarding median glucose or glycated hemoglobin (HbA1c) levels (P = 0.465 and 0.932, respectively). Moreover, levels of glucose (odds ratio (OR) 0.928, confidence interval (CI) 0.763-1.13, P = 0.457) and HbA1c (OR 0.89, CI 0.63-1.26, P = 0.507) were not risk factors for increased mortality among diabetic patients. Conclusion We demonstrated the association between DM and increased CRE mortality regardless of the level of glycemic control. This study demonstrates the interaction between communicable and non-communicable diseases.

Outer membrane vesicles mediating horizontal transfer of the epidemic bla OXA-232 carbapenemase gene among Enterobacterales

ABSTRACT OXA-232 is one of the most common OXA-48-like carbapenemase derivatives and is widely disseminated in nosocomial settings across countries. The bla OXA-232 gene is located on a 6-kb non-conjugative ColKP3-type plasmid, while the dissemination of bla OXA-232 into different Enterobacterales species and the polyclonal dissemination of OXA-232-producing K. pneumoniae revealed the horizontal transfer of bla OXA-232. However, it’s still unclear how this non-conjugative ColKP3 plasmid could facilitate the mobilization of bla OXA-232. Here, we observed the in vivo intraspecies transfer of bla OXA-232 during a nosocomial outbreak of OXA-232-producing K. pneumoniae. We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of bla OXA-232 among Enterobacterales. In contrast, for the most prevalent carbapenemase genes, including bla KPC-2 and bla NDM-1, though the presence of carbapenemase genes and plasmid backbones in the vesicular lumen was observed, OMVs couldn’t promote effective transformation, probably due to the low copy number of plasmids in clinical isolates and the low number of plasmids loaded into vesicles. Conjugation assay revealed that the epidemic IncX3 NDM-1 and IncFII(pHN7A8)/IncR KPC-2 plasmids were conjugative and could be horizontally transferred via independent conjugation or with the help of a co-existent conjugative plasmid. For the large-size and low-copy number conjugative plasmids carrying carbapenemase genes, OMVs-mediated gene exchange may only serve as an alternative pathway for horizontal transfer. In conclusion, diverse mobilization strategies were employed by plasmids harbouring carbapenemase genes, and plasmids display a proper choice of mobility pathway due to their individual properties.

Carbapenem-Resistant Enterobacterales Infections and Their Association with Rectal Colonization

Objectives: Carbapenem-resistant Enterobacterales (CRE) infections have limited treatment options, and these infections are associated with high mortality rates. Asymptomatic carriers colonized with CRE contribute to the spread of CRE in hospitals. It was aimed to determine the frequency of CRE isolates detected in our center, carbapenemase ratios in these strains, carbapenemase genes, antibiotic resistance profiles, rectal CRE colonization rates and to evaluate various clinical features of CRE infections.
 Methods: Enterobacterales species isolated from various specimens and Enterobacterales species isolated from rectal swab specimens sent for colonization screening were examined. Patients with CRE colonization in rectal swab samples were examined for the development of CRE infection at a later time. CRE isolates were examined for carbapenemase production and the presence of carbapenemase gene.
 Results: 12,721 Enterobacterales (10,161 E. coli and 2395 K. pneumoniae, 165 Citrobacter) isolates were examined. Carbapenem resistance was detected in 10.3% of these strains. CRE was detected in 4.7% of 15695 rectal swab samples evaluated for colonization. In 23.4% of the patients with CRE colonization, CRE growth was detected in other samples besides the rectal swab in the later period. It was observed that CRE infections developed on average 21 days after colonization.
 Conclusion: CRE infections have started to emerge as a factor not only in hospitalized patients but also in community-acquired infections. Our study also showed that CRE colonization could be a significant risk factor for the development of infection. Therefore, early screening detection to detect colonization can help prevent or limit CRE infections with appropriate isolation methods.

Antimicrobial Resistance, Genetic Lineages, and Biofilm Formation in Pseudomonas aeruginosa Isolated from Human Infections: An Emerging One Health Concern.

Pseudomonas aeruginosa (PA) is a leading nosocomial pathogen and has great versatility due to a complex interplay between antimicrobial resistance and virulence factors. PA has also turned into one the most relevant model organisms for the study of biofilm-associated infections. The objective of the study focused on analyzing the antimicrobial susceptibility, resistance genes, virulence factors, and biofilm formation ability of thirty-two isolates of PA. PA isolates were characterized by the following analyses: susceptibility to 12 antimicrobial agents, the presence of resistance genes and virulence factors in PCR assays, and the quantification of biofilm production as evaluated by two distinct assays. Selected PA isolates were analyzed through multilocus sequence typing (MLST). Thirty PA isolates have a multi-resistant phenotype, and most of the isolates showed high levels of resistance to the tested antibiotics. Carbapenems showed the highest prevalence of resistance. Various virulence factors were detected and, for the quantification of biofilm production, the effectiveness of different methods was assessed. The microtiter plate method showed the highest accuracy and reproducibility for detecting biofilm-producing bacteria. MLST revealed four distinct sequence types (STs) in clinical PA, with three of them considered high-risk clones of PA, namely ST175, ST235, and ST244. These clones are associated with multidrug resistance and are prevalent in hospitals worldwide. Overall, the study highlights the high prevalence of antibiotic resistance, the presence of carbapenemase genes, the diversity of virulence factors, and the importance of biofilm formation in PA clinical isolates. Understanding these factors is crucial for effective infection control measures and the development of targeted treatment strategies.

Emergence of carbapenem-resistant Enterobacter hormaechei ST93 plasmids co-harbouring blaNDM-1, blaKPC-2, and mcr-9 in bloodstream infection

We isolated a strain of Enterobacter hormaechei, ECC2783, co-harbouring blaNDM-1, blaKPC-2 and mcr-9 plasmids from a bloodstream infection and investigated its biological features. The presence of carbapenemase genes and mcr-9 was confirmed by polymerase chain reaction amplification. Whole genome sequencing and genomic analysis were performed on ECC2783. Experiments assessing the conjugation and stability of plasmids carrying the carbapenemase gene were performed. We also performed a colistin resistance induction experiment and studied the fitness cost of transconjugants. ECC2783 has an extensive drug resistance phenotype. Multilocus sequence typing analysis results showed that ECC2783 belongs to sequence type 93. Bioinformatics analysis confirmed that ECC2783 has four plasmids, of which pECC2783_a, carrying mcr-9, is the IncHI2 type, and pECC2783_c, carrying blaNDM-1, is the IncX3 type. pECC2783_d, carrying blaKPC-2, is an unclassified type. We successfully obtained two transconjugants (J53/ECC2783_1, carrying blaNDM-1, and J53/ECC2783_2, carrying blaKPC-2 and blaNDM-1). There was no statistically significant difference in the relative growth rate between J53 and J53/ECC2783_2. For the first time, we isolated carbapenem-resistant E. hormaechei plasmids co-harbouring blaNDM-1, blaKPC-2, and mcr-9 from a patient with a blood stream infection. This isolate has a survival advantage in a hospital environment, and its clinical monitoring should be strengthened.

The Prevalence of Colonization with Carbapenem-resistant Enterobacteriaceae, Escherichia coli, Klebsiella and Enterobacter, and Related Risk Factors in Children

Background: Carbapenems are broad-spectrum antibiotics used to treat the family of gram-negative Enterobacteriaceae, especially those that are resistant to first-line antibiotics. Because these drugs are usually prescribed as the last line of treatment, resistance to these antibiotics carries irreparable risks to treatment systems, and screening high-risk individuals in medical centers and using infection control measures are critical strategies for eliminating them. Objectives: We investigated the prevalence of colonization of different strains of Enterobacteriaceae, Klebsiella, Enterobacter, and Escherichia coli and their risk factors in hospitalized children. Methods: In this descriptive cross-sectional study, stool samples were taken from patients during the first 48 hours of hospitalization in a tertiary children’s hospital and were cultured on Makcanki culture medium or EMB. Cultured Enterobacteriaceae samples were transferred to Müller-Hinton agar medium, and their antibiotic susceptibility was evaluated with meropenem and imipenem discs by disc diffusion method. In the next step, five common carbapenemase genes, including (VIM, IMP, OXA-48, NDM-1, and SPM-1) were examined by PCR method and reported accordingly. Results: Two hundred and ninety-five stool samples were examined, of which 242 (82%) samples were cultured positively with Enterobacteriaceae. The prevalence of carbapenem resistance was reported to be 37% among 295 samples using the phenotypic method. Resistance rates were high in patients with a history of antibiotic use, with frequent hospitalizations (more than two episodes in the last six months), and in patients with an underlying disease) malignancy, GI diseases, immunodeficiency, neurologic diseases such as cerebral palsy and epilepsy, endocrine diseases. Most of the genes found were OXA-48, followed by IMP and VIM. NDM-1 was found in 3 samples, and SPM was not found in any of the samples. In 13% of resistant samples, more than one carbapenemase gene was found. Conclusions: The results of this study showed that the frequency of carbapenem resistance in stools colonized with Enterobacteriaceae is high in our patients. On the other hand, the presence of carbapenemase genes in these bacteria, which are located on the plasmids that can be rapidly spread in the hospital environment, is an alarm for the hospital infection control committee to take preventive measures in order to prevent the spread of these bacteria in the hospital.

643. Cefiderocol In vitro Activity Against Molecularly Characterized Pseudomonas aeruginosa and Acinetobacter baumannii-calcoaceticus complex Clinical Isolates Causing Infection in United States Hospitals (2020-2021)

Abstract Background Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin with activity against Gram-negative bacteria. CFDC and comparator activities were analyzed against resistant and molecularly characterized P. aeruginosa (PSA) and A. baumannii-calcoaceticus complex (ACB) as part of the SENTRY Antimicrobial Surveillance Program. Methods 2,241 PSA and 682 ACB were consecutively collected from 63 US sites in 2020-2021. Susceptibility testing was performed by broth microdilution and CFDC testing used iron-depleted media. FDA and CLSI breakpoints were used for CFDC. CLSI criteria were applied to comparators, except for imipenem-relebactam (IMR) that used FDA breakpoints. Isolates with a resistance phenotype to ≥ 3 classes were defined as multidrug resistant (MDR). ACB and PSA with imipenem and/or meropenem (MER) MIC ≥4 μg/mL or ceftazidime (CAZ) and/or cefepime MIC ≥ 16 μg/mL were subjected to next-generation genome sequencing for screening of acquired carbapenemase genes. Results 31.6% (709/2,241) and 16.9% (379/2,241) of PSA met the MIC screening criteria and showed an MDR phenotype, respectively (Table). Carbapenemase genes were detected in 6 (< 1%) PSA. CFDC had similar MIC against PSA that did not (MIC50/90, 0.06/0.25 μg/mL) and did (MIC50/90, 0.12/0.5 μg/mL) meet the MIC screening criteria. CFDC also had similar MIC50 values (0.12 μg/mL) against the MDR and carbapenemase-positive PSA populations, whereas other agents had compromised activity. 42.5% (290/682) of ACB met the MIC screening criteria, while 35.9% (245/682) had an MDR phenotype, and 27.3% carried carbapenemase genes (all OXA carbapenemase, except for 2 blaNDM-1). In general, CFDC, IMR, meropenem-vaborbactam, MER, ceftazidime-avibactam, and CAZ had activity against ACB that did not meet the MIC screening criteria, but only CFDC (MIC50/90, 0.25-0.5/2 μg/mL) was active against the resistant ACB subsets. Conclusion Many PSA showed a resistance phenotype but acquired carbapenemase genes remained rare in this subset. In contrast, resistance and presence of carbapenemase genes were high in ACB. CFDC showed potent activity against PSA and ACB subsets in US hospitals, including across resistant and molecularly characterized subsets, where treatment options were limited. Disclosures Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support Cory Hubler, BS, Melinta: Grant/Research Support|Shionogi: Grant/Research Support Valerie Kantro, BA, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.

Clinical Outcomes Associated with Co-infection of Carbapenem-Resistant Enterobacterales and other Multidrug-Resistant Organisms

Infections with carbapenem-resistant Enterobacterales (CRE) are associated with increased risk of death. Polymicrobial infections with antimicrobial-resistance may add to the burden of clinical care and patients' clinical prognosis. To examine the impact of CRE co-infection with other multi-drug resistant organisms (MDRO) on patient clinical outcomes. A retrospective observational study was conducted to compare the clinical outcomes of CRE patients who were co-infected with carbapenem-resistant Pseudomonas aeruginosa (CRPA), multidrug-resistant Acinetobacter baumannii (MDRA) and Methicillin-resistant Staphylococcus aureus (MRSA). A total of 224 CRPA and 209 MDRA co-infections with CRE were identified from 4,236 cases from 2015-2020. The overall 90-day all-cause mortality was 21.6% but increased to 35.0% and 33.5% among patients who were co-infected with CRPA and MDRA, respectively. The odds of all-cause mortality among CRE patients who were co-infected with CRPA was twice that of patients identified with CRE alone [adjusted odds ratio (AOR)= 2.02, 95% confidence interval (CI): 1.18-3.46]. Further, the odds of all-cause mortality among CRE patients who were concomitantly identified with MRSA was more than twice that of patients who were not identified with MRSA [AOR= 2.16, 95%CI:1.31-3.56]. The clinical outcome of patients with CRE did not differ significantly depending on the presence of carbapenemase genes. The results show that CRPA and CRE co-infections have synergistic effects on clinical outcomes. Further investigation is necessary to understand the mechanism. Screening high risk patients for concomitant antimicrobial-resistant infections may have a significant clinical impact, including effective therapies, antibiotic stewardship, and infection control policies.

Analysis of In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Gram-Negative Bacilli by Broth Microdilution and Disk Diffusion Method: A Single-Center Study in Odisha, India

IntroductionCefiderocol (CFDC), a novel semi-synthetic siderophore cephalosporin has been developed to combat the menace of infections caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) including Carbapenem-resistant Enterobacterales (CRE) and Carbapenem-resistant Nonfermenting Gram-negative bacilli (CR-NFGNB).MethodsWe determined the in vitro activity of CFDC against a contemporary collection of 503 CR-GNB isolates by the reference broth microdilution method (BMD) using Iron depleted cation adjusted Mueller-Hinton broth (ID-CAMHB). Performance of CFDC disk diffusion (DD) was evaluated against the reference BMD, as an alternative convenient testing method. Molecular characterization of carbapenemase in CR-GNB was performed by PCR targeting blaNDM-1, blaOXA-48like alleles, blaKPC, blaIMP, and, blaVIM. Minimum inhibitory concentration (MIC) distribution of CFDC in CR-GNB harbouring different carbapenemase enzymes was also analyzed.ResultsIn our study, 81.7% (411/503) of CR-GNB isolates [81.3%, (278/342) CRE and 82.6% (133/161) CR-NFGNB] were susceptible to CFDC (p>0.05). Categorical agreement (CA) of DD ranged from 79.8% to 87.5%, Minor error (mE) ranged from 0 to 14%, Major error (ME) ranged from 0 to 3.5%, and Very Major error (VME) ranged from 0 to 12.5% with variations by species tested. Overall CFDC MIC50 and MIC90 values of CR-GNB isolates without any carbapenemase genes were higher as compared to those with the presence of carbapenemase genes (4 µg/mL and 128 µg/mL versus 2 µg/mL and 16 µg/mL respectively).DiscussionCFDC is not yet available for clinical use in India. Hence, multicentric studies are the need of the hour in India for standardization of CFDC susceptibility using disks and CAMHB from different manufacturers as well as understanding mechanisms of high MIC values.

Genetic Diversity, Carbapenem Resistance Genes, and Biofilm Formation in UPEC Isolated from Patients with Catheter-Associated Urinary Tract Infection in North of Iran.

Background Infections due to carbapenem-resistant Enterobacteriaceae (CRE) are associated in patients with urinary catheters alarming rate of emergency status. The aim of this study is to investigate the molecular causes of carbapenem resistance among UPEC as well as antimicrobial resistance trends. Additionally, the potential of isolates to produce biofilms, in addition to their clonal and genetic diversity, was investigated. Material and Methods. A cross-sectional study was accomplished on a collection of 76 non-duplicate UPEC isolates obtained from CAUTIs from May 2021 to September 2021. The modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) test was performed for the detection of carbapenemase and metallo-beta-lactamase activity. Also, the presence of carbapenemase genes was determined using PCR assays. In 96-well microtiter plates, biofilm development was evaluated. ERIC-PCR was used to investigate the clonal and genetic variety of isolates. Results A total of 76 confirmed UPEC isolates were obtained from patients mentioned to teaching hospitals in Babol, Iran. The results of antibiotic susceptibility testing revealed a high rate of antibiotic resistance against nalidixic acid (81.6%) and trimethoprim-sulfamethoxazole (80.3%). Among UPEC isolates, 63.2% and 13.2% of UPEC isolates were positive for MBL production. The frequencies of the studied genes are in order of blaNDM (14.5%), blaOXA-23 (2.6%), and blaOXA-48 (2.6%). Forty-two isolates (55.3%) were positive for biofilm formation. ERIC-PCR revealed that UPEC isolates could be categorized into nine clusters A-I and five isolates were categorized as a singleton. Conclusion The high prevalence of MDR and carbapenemase-producing isolates among the UPEC strain in this investigation is concerning. Moreover, the blaNDM was the most frequent cause of producing metallo-beta-lactamase and carbapenemase. Also, analysis revealed a partial genetic similarity among the studied isolates, indicating that the same UPEC clones may have spread to other hospital units.

Molecular characterization of carbapenem-resistant Pseudomonas aeruginosa isolated from four medical centres in Iran.

Understanding the mechanisms of antibiotic resistance is important for designing new therapeutic options and controlling resistant strains. The goal of this study was to look at the molecular epidemiology and mechanisms of resistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from Tabriz, Iran. One hundred and forty P. aeruginosa were isolated and antibiotic susceptibility patterns were determined. Overproduction of AmpC and efflux pumps were discovered using phenotypic techniques. Polymerase chain reaction (PCR) was used to determine the presence of carbapenemase-encoding genes. In addition, the expressions of OprD and efflux pumps were evaluated by the Real-Time PCR. Random amplified polymorphic DNA typing (RAPD) was performed for genotyping. Among 140 P. aeruginosa isolates, 74 (52.8%) were screened as CRPA. Overexpression of efflux systems was observed in 81% of isolates, followed by decreased expression of OprD (62.2%), presence of carbapenemase genes (14.8%), and overproduction of AmpC (13.5%). In most isolates, carbapenem resistance was multifactorial (60.8%). According to our results, the prevalence of CRPA is at alarming levels. Overexpression of efflux systems was the most common mechanism of carbapenem resistance. Most isolates may originate in patients themselves, but cross-infection is possible. Therefore, we suggest a pattern shift in the strategy of CRPA in our setting.

Comparison of in Vitro Antimicrobial Efficacy of Ceftolozane-Tazobactam and Ceftazidime-Avibactam Combination Against Carbapenem-Resistant Enterobacteriaceae Species Isolated from Various Clinical Specimens

Objective: The increase in resistant Gram-negative bacteria is a major concern and has led to difficulties in the treatment of infections. The aim of this study was to compare the in vitro efficacy of CLZ-TAZ and CAZ-AVB combinations against carbapenem-resistant Enterobacteriaceae strains.
 
 Methods: 80 carbapenem-resistant Enterobacteriaceae species isolated from various samples sent to our laboratory were included in the study. Identification and antimicrobial susceptibility of strains were performed using automated systems. The presence of carbapenemases in all isolates was tested using the CarbaNP test and the carbapenem inactivation method. The presence of carbapenemase genes was tested by multiplex PCR.
 
 Results: The presence of carbapenemases was detected in 60 % E. coli isolates and in 78.5% K. pneumoniae isolates via phenotypic tests. OXA-48 enzyme was found in 73.7% of isolates containing carbapenemase. The second most common enzyme was NDM.The assessment of the efficacy of the β-lactam/β-lactamase inhibitor combinations against CRE isolates revealed that the activity of CAZ-AVB (77%) was higher than CLZ-TAZ (48%).
 
 Conclusion: Our findings suggest that CAZ-AVB and CLZ-TAZ may be promising in the treatment of infections caused by CRE strains. Sensitivity rates were higher with ceftazidime-avibactam than with ceftolozane-tazobactam. The data obtained in this study will contribute to the clinical use of these agents in our country.

A retrospective study on molecular epidemiology trends of carbapenem resistant Enterobacteriaceae in a teaching hospital in Malaysia.

BackgroundCarbapenem resistant Enterobacteriaceae (CRE) has rapidly disseminated worldwide and has become a global threat to the healthcare system due to its resistance towards “last line” antibiotics. This study aimed to investigate the prevalence of CRE and the resistance mechanism as well as the risk factors associated with in-hospital mortality.MethodsA total of 168 CRE strains isolated from a tertiary teaching hospital from 2014–2015 were included in this study. The presence of carbapenemase genes and minimum inhibitory concentration of imipenem, meropenem and colistin were investigated. All carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains were characterised by PFGE. The risk factors of patients infected by CRE associated with in-hospital mortality were determined statistically.ResultsThe predominant CRE species isolated was K. pneumoniae. The carbapenemases detected were blaOXA-48, blaOXA-232, blaVIM and blaNDM of which blaOXA-48 was the predominant carbapenemase detected among 168 CRE strains. A total of 40 CRE strains harboured two different carbapenemase genes. A total of seven clusters and 48 pulsotypes were identified among 140 CRKp strains. A predominant pulsotype responsible for the transmission from 2014 to 2015 was identified. Univariate statistical analysis identified that the period between CRE isolation and start of appropriate therapy of more than 3 days was statistically associated with in-hospital mortality.

Investigation of major carbapenemase genes in ESBL-producing Escherichia coli and Klebsiella pneumoniae strains isolated from raw milk in Black Sea region of Turkey

The prevalence of blaKPC, blaNDM, blaOXA-48, blaIMP and blaVIM genes and phenotypic carbapenemase production was investigated in ESBL-producing Enterobacteriaceae strains isolated from 150 raw cow milk samples collected from the Black Sea region of Turkey. A total of 47 phenotypic ESBL producers, 41 Escherichia coli and six Klebsiella pneumoniae, were investigated in terms of carbapenemase activity. The presence of carbapenemase genes was investigated using five single PCR assays. Seven of the 47 isolates were found resistant to imipenem and/or meropenem. PCR assays revealed that 33 of the 47 isolates were found to harbour at least one of the analysed carbapenemase genes. For blaKPC, blaOXA-48, blaIMP and blaVIM genes, BLAST search indicated 97.9%, 97.2%, 97.1% and 97% nucleotide identity to corresponding sequences, respectively. Carbapenemase-producing E. coli and K. pneumoniae were found higher in milk compared with studies conducted worldwide and carbapenemase production was associated with blaOXA-48 and blaKPC.

Using Whole Genome Sequencing to Investigate a Mock-Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in Real-Time.

Healthcare associated infections due to carbapenem-resistant Klebsiella pneumoniae (CRKP) are a major concern in Portuguese hospitals. Whole genome sequencing (WGS) can improve infection control, but this practice is not routinely used by hospital clinical laboratories in Portugal. We simulated the investigation of a CRKP outbreak based on WGS, with the aim of determining, in the minimum possible time, genetic relatedness between CRKP clinical and environmental isolates. Ten CRKP clinical isolates routinely obtained in the hospital laboratory were used. Forty environmental samples - from sinks and sink drains of ward rooms - were collected. Environmental samples were plated on selective media and presumptive CRKP colonies were isolated. Total DNA was extracted from all putative CRKP isolates and sequenced. Clonal relatedness was determined by multi-locus sequence typing and core genome single nucleotide polymorphism analysis; the presence of carbapenemase genes was evaluated. Clinical isolates were characterized in 48 hours: eight strains were confirmed as CRKP, of which six were of ST13 and carried blaKPC-3. Environmental samples results were obtained in six days: eight CRKP were isolated from which five were of ST13 and carried blaKPC-3. Clinical and environmental ST13 isolates were highly related: ten (of 11) isolates differed from each other in < 0.001% of 2 172 367 core nucleotides. WGS can be used as a high-resolution effective tool to investigate healthcare associated infections and track routes of dissemination in real-time. In Portugal, routine use of WGS to improve infection control could thrive through collaborative initiatives between hospitals and research institutes.

Evaluation of the BD Phoenix CPO detect panel for prediction of Ambler class carbapenemases

Rapid detection of carbapenemases as a cause of resistance is beneficial for infection control and antimicrobial therapy. The BD Phoenix NMIC-502 panel and CPO detect test identifies presence of carbapenemases in Enterobacterales such as Klebsiella pneumoniae and assigns them to Ambler classes. To evaluate the performance of the CPO detect panel, we employed a European collection of 1222 K. pneumoniae including carbapenem non-susceptible and susceptible clinical isolates from 26 countries, for which draft genomes were available after Illumina sequencing and the presence of carbapenemase genes had been identified by ARIBA gene calling. The CPO panel detected 488 out of 494 carbapenemase-encoding isolates as positive and six as negative. One-hundred and two isolates were tested positive for carbapenemase in the absence of any carbapenemase gene. The CPO panel identified 229 out of 230 KPC-positive isolates as carbapenemase producing and classified 62 of these as class A enzyme. Similarly, the CPO panel correctly specified 167 of 182 as class D. Regarding metallo-beta-lactamases, the CPO panel assigned 78 of 90 MBL positive isolates to class B enzymes. The sensitivity of the CPO panel in detecting carbapenemase activity was 99.5%, 97.7% and 98.3% for class A, B and D enzymes, respectively. The sensitivity in assignation to Ambler class A, B and D was 27%, 86% and 91%, respectively. An overall sensitivity of 98.8% and specificity of 86% in unclassified detection of carbapenemases was observed, with frequent false positive detection of carbapenemase producing organisms, thus rendering further confirmatory tests necessary.

Silent spread of mobile colistin resistance gene mcr-9.1 on IncHI2 ‘superplasmids’ in clinical carbapenem-resistant Enterobacterales

Objectivesmcr-9.1 is a newly described mobile colistin resistance gene. We have noted its presence in multiple species of carbapenem-resistant Enterobacterales (CRE) from our institution. We aimed to determine the clinical features, genomic context and phenotypic impact of mcr-9.1 carriage in a series of patients between 2010 and 2019. MethodsWe identified 32 patients with mcr-9.1-carrying CRE isolates (mCRE) and collected demographic, antimicrobial exposure and infection data. Whole-genome sequencing (including short and long reads) was performed on 32 isolates. We assessed sequence similarity of mcr-9.1-harbouring plasmids, then compared our findings with plasmids for which sequence data were publicly available. ResultsThere was no colistin exposure in patients prior to isolation of mCRE. mcr-9.1 was identified on IncHI2 plasmids across four different bacterial species and was co-located with blaIMP-4 in 23/30 plasmids studied. mCRE isolates did not demonstrate phenotypic colistin resistance, either at baseline or following sublethal colistin exposure, thus showing that mcr-9.1 alone is not sufficient for resistance. Publicly available sequence data indicated the presence of carbapenemase genes in 236/619 mcr-9.1-carrying genomes (38%). IncHI2 plasmids carrying mcr-9.1 and carbapenemase genes were detected in genomes from North America, Europe, North Africa, Asia and Oceania. ConclusionsSpread of mcr-9.1 in CRE from our institution was driven by IncHI2 ‘superplasmids’, so termed because of their large size and their prolific carriage of resistance determinants. These were also detected in global CRE genomes. Phenotypic colistin resistance was not detected in our isolates but remains to be determined from global mCRE.

Cefoperazone/sulbactam: New composites against multiresistant gram negative bacteria?

Sulbactam, a class A β-lactamase inhibitor, added to cefoperazone either at a fixed 8mg/L level of sulbactam or at a level of fixed cefoperazone: sulbactam ratio (2:1) would constitute a combination form of cefoperazone/sulbactam, which has better activities against Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii than cefoperazone alone. Cefoperazone/sulbactam (1:1 or 1:2) has greater in-vitro activity against most multidrug-resistant organisms (ESBL- and AmpC-producing Enterobacteriaceae and carbapenem-resistant A. baumannii except for carbapenem-resistant P. aeruginosa) than a 2:1 ratio. However, increased sulbactam concentration may induce AmpC production. Besides, sulbactam concentration might not be readily achievable in serum if the susceptibility rates were defined by the breakpoints of higher sulbactam composites, such as ≤16/16 (1:1) or 16/32 (1:2) mg/L. Carbapenemases (KPC-, OXA-type enzymes and metallo-β-lactamases) can't be inhibited by sulbactam. Some in-vitro studies showed that increasing sulbactam composites of cefoperazone/sulbactam had no effect on carbapenem-resistant P. aeruginosa, suggesting the presence of carbapenemases or AmpC overproduction that could not be overcome by increasing sulbactam levels to recover cefoperazone activity. Sulbactam alone has good intrinsic activity against carbapenem-resistant Acinetobacter strains sometimes even in the presence of carbapenemase genes, suggesting unsteady levels of carbapenemases. In conclusion, appropriate composites of cefoperazone and β-lactamase inhibitor sulbactam may expand the clinical use if the pharmacokinetic optimization could be achieved in the human serum.