Modified Carbapenem Inactivation Method Research Articles

The Increased Prevalence of rmpA Gene in Klebsiella pneumoniae Isolates Coharboring blaNDM and blaOXA-48-like Genes.

The emergence of carbapenemase-producing Klebsiella pneumoniae poses a substantial risk to public health. It is essential to comprehend the influence of carbapenemase on the virulence characteristics of K. pneumoniae in order to devise successful strategies for combating these infections. In this study, we explored the distribution disparity of virulence determinants between carbapenemase-producing (CP-Kp, n = 52) and carbapenemase-nonproducing (CN-Kp, n = 43) isolates. The presence of carbapenemases was detected via the modified carbapenem inactivation method and confirmed by PCR. The New Delhi metallo-β-lactamase (blaNDM) and Oxacillinase-48-like (blaOXA-48-like) genes were the most prevalent (94.23% and 76.92%, respectively) in CP-Kp isolates. Coexistence of blaNDM and blaOXA-48-like was observed in 71.15% of isolates, whereas 5.77% coharbored blaNDM and blaKPC. PCR analysis revealed the presence of several virulence genes, including adhesins (fimH, 92.63%, mrkD, 97.89%), capsule-associated virulence (uge, 90.53%), the K2 capsule serotype (k2, 6.32%), the iron acquisition system (kfu, 23.16%), and the regulator of mucoid phenotype (rmpA, 28.42%). A significantly higher prevalence of rmpA was detected in the CP-Kp compared with the CN-Kp (24/52 vs. 3/43, p < 0.0001), indicating a potential association between rmpA and carbapenemase acquisition. In addition, the majority of rmpA (22/24) positive isolates in the CP-Kp isolates coharbored blaNDM and either blaOXA-48-like or blaKPC.

Molecular detection of blaNDM and blaOXA-48 genes in Carbapenem-Resistant Klebsiella pneumoniae isolates from a tertiary care hospital

BackgroundThe incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is increasing globally. In India, around 65% of K. pneumoniae isolates are resistant to carbapenem antibiotics. The blaNDM is the predominant carbapenem-resistant gene in CRKP isolates. However, blaOXA-48 has also been reported to be increasing in recent days. MethodsK. pneumoniae strains isolated from the clinical specimens of patients attending a tertiary care hospital in Hyderabad from June 2021 to May 2022 were included in this study. Resistance to carbapenems and other antibiotics was screened using Kirby-Bauer’s disc diffusion method. Phenotypic tests such as the Carbapenemase Nordmann Poirel (CarbaNP) test, Modified Carbapenem Inactivation Method (mCIM), and EDTA Carbapenem Inactivation Method (eCIM) were used to detect the carbapenemase producers. The genes responsible for carbapenemase production were detected by the real-time polymerase chain reaction (RT-PCR) method. ResultsOut of 1265 K. pneumoniae strains isolated, 241 (19 %) isolates showed resistance to any one of the carbapenem antibiotics tested. Phenotypic tests such as CarbaNP, mCIM, and eCIM revealed that 94.6 %, 97.1 %, and 95.4 % of CRKP isolates were carbapenemase producers, respectively. About 66.4 % of CRKP isolates harbored blaNDM, 17.4 % harbored blaOXA-48, and 16.2 % harbored both blaNDM and OXA-48 genes. None of the isolates tested positive for blaKPC, blaVIM, and blaIMP genes. ConclusionIn this study, blaNDM is the most prevalent carbapenemase gene in CRKP isolates. Furthermore, blaOXA-48 and the co-existence of both blaNDM and blaOXA-48 genes were also found. CRKP is emerging as a serious threat among drug-resistant bacterial pathogens which would complicate the treatment of bacterial infections with available antibiotics.

Phenotypic detection of carbapenem-resistant Enterobacterales in clinical isolates at a tertiary care hospital

Abstract Background: In the past decade, there has been a global emergence of carbapenem-resistant Gram-negative bacilli, especially Enterobacterales. Carbapenem resistance is attributed to the ability of the bacteria to produce carbapenemases. The aim of the study is to detect carbapenem-resistant Enterobacterales (CRE) in different clinical isolates and study carbapenemase production by phenotypic methods in CRE. Materials and Methods: A total of 379 Enterobacterales were isolated from different clinical samples from patients attending outpatient departments and admitted in wards and intensive care units (ICUs). They were tested for carbapenem resistance by Kirby–Bauer disk diffusion method and then tested for carbapenemase production by ethylenediaminetetraacetic acid (EDTA)–disk synergy test and Modified Carbapenem Inactivation Method (mCIM). Results: This study was conducted from February 2021 to August 2022. Out of 379 Enterobacterales, 70 (18.47%) were CRE, out of which maximum carbapenem resistance of 23.53% was shown by Klebsiella pneumoniae isolates. The maximum carbapenem resistance was seen in the age group of 16–45 years and the most number of CRE isolates were from ICUs. Phenotypic test results indicated that 54.28% (38/70) of isolates were positive for carbapenemase production by either of the phenotypic methods. Conclusion: About one-fifth of the Enterobacterales isolates were carbapenem resistant. This study highlights the use of phenotypic methods to detect carbapenemase production in CRE, which is responsible for multidrug resistance. This information is relevant for surveillance, to implement infection prevention and control practices and antibiotic policies.

Prevalence and characteristics of ertapenem-mono-resistant isolates among carbapenem-resistant Enterobacterales in China

ABSTRACT Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to β-lactams, β-lactam/β-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum β-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.

Phenotypic and Molecular Detection of Carbapenemase-Producing Klebsiella pneumoniae Isolated from Patients Admitted to Teaching Hospitals in Shiraz, Iran

Background: Carbapenem-resistant Klebsiella pneumoniae (Cr-KPN) poses a significant global public health challenge. Objectives: This study aimed to investigate the prevalence and expression levels of carbapenemase-encoding genes in Cr-KPN isolated from patients admitted to teaching hospitals in Shiraz, Iran. Methods: A total of 671 distinct clinical samples were collected from two teaching hospitals in Shiraz. Initial identification and final confirmation of K. pneumoniae isolates were carried out using conventional biochemical tests and PCR assays, respectively. The detection of carbapenemase-producing K. pneumoniae, both phenotypically and genotypically, was performed through modified carbapenem inactivation methods (mCIM) and multiplex PCR assays. Real-time PCR was utilized to assess the expression levels of carbapenemase-encoding genes. Results: The overall frequency of K. pneumoniae strains was 14.9% (n = 100/671). mCIM indicated that 26% of K. pneumoniae isolates exhibited carbapenemase production. Furthermore, 24% and 17% of K. pneumoniae isolates demonstrated resistance to imipenem and meropenem, respectively. The blaIMI/IMP gene was detected in 91% of the isolates. Among imipenem-resistant isolates, 62.5% tested positive for the blaOXA-48 gene. Additionally, 29.4%, 76.5%, and 11.8% of meropenem-resistant isolates were positive for the blaKPC, blaOXA-48, and blaNDM genes, respectively. Real-time PCR analysis revealed increased expression levels of blaKPC (1.66-fold), blaOXA-48 (7.30-fold), blaNDM (4.22-fold), and blaIMI/NMC (2.39-fold) genes in resistant isolates when exposed to imipenem. Conclusions: These findings underscore the significance of establishing active surveillance networks to monitor and track the dissemination of carbapenemase-producing K. pneumoniae, which presents a global public health threat.

EMERGENCE OF CEFTAZIDIME-AVIBACTAM RESISTANCE IN ENTERO-BACTERALES AND PSEUDOMONAS AERUGINOSA

Objective: The objective of this study is to determine the emergence of resistance to Ceftazidime-Avibactam (CAZ-AVI) by Enterobacterales and Pseudomonas aeruginosa in clinical isolates. Material and Methods: In this cross-sectional study (6-months) March-August 2022 Carbapenem resistant Enterobacterales were tested for Ceftazidime-Avibactam (30/20 µg, Oxoid Pvt Ltd) using Disk diffusion technique and enzymes were identified in resistant strains by Carbapenem Inactivation Method (mCIM, eCIM) as per CLSI M100 Guidelines 2022. Results: CAZ-AVI effectiveness has greatly decreased among Enterobacterales and P. aeruginosa isolates in recent past. Antimicrobial susceptibility testing results were interpreted using CLSI M100 document. Resistance against CAZ-AVI in Enterobacterales was found to be 80.8 % in E. coli and 72.1% in Klebsiella pneumonia isolates. This higher emergence is associated with CRE isolates majorly comprising MBLs in our country. Moreover, it has been observed that Metallo- β-lactamases mediated enzyme resistance is one of the major resistance patterns followed by serine carbapenemases. Conclusion: The high frequency of resistance 77% was observed against CAZ-AVI in CRE and in CRPA, the resistance is 80.1% respectively. In our country this tremendously increase in CAZ-AVI resistance is attributed to the existence of NDM in the region. Keywords: Carbapenem Resistant Enterobacterales (CRE), Metallo beta lactamases (MBLs), Carbapenem Resistant Pseudomonas aeruginosa (CRPA) Ceftazidime-Avibactam (CAZ-AVI), Modified Carbapenem Inactivation Method (mCIM), EDTA-modified Carbapenem Inactivation Method(eCIM), Multiple Drug Resistance (MDR)

Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Detection of hypervirulent and classical type of Klebsiella pneumoniae and screening their resistant properties in Erbil city

Background and objective: Klebsiella pneumoniaei s an opportunistic pathogenic bacterium and is considered one of the main causes of nosocomial infection. Hypervirulent K. pneumoniae (hvKp) has emerged as a pathogen of global importance. The current study aimed to determine Extended-spectrum beta-lactamase, carbapenem resistance, and serum bactericidal effect among the clinical isolates and to find the relationship of the above features with antimicrobial resistance. Methods: A total of 90 K. pneumoniae isolates were collected from different clinical specimens. Isolates were diagnosed using routine bacteriological methods and VITEK 2 compact system. Several phenotypic tests including string test, serum resistant, ESBL test, and Modified Carbapenem inactivation method were performed. The antibiotic resistance pattern was compared among ESBL-positive, carbapenem-resistant in both Hypervirulent (hvKp) and classical K. pneumoniae (cKp) isolates. Results: The results revealed that among 90 isolates, 56.7% of the isolates were of classical K. pneumoniae (cKp) type within which 70.6% of them were ESBL positive, 37.3% of them were resistant to carbapenem, and 51% were resistant to serum bactericidal activity. On the other hand, 43.3% were of hvKp type within which 61.5% of them were ESBL positive, 30.8% were carbapenem-resistant and all of the hvKp were resistant to human serum. The rate of antibiotic-resistant among cKp was higher than hvKp isolates. Conclusion: In this study, classical strains were more resistant to antibiotics and the rates of ESBL and Carbapenem resistance were higher compared to hvKp strains. but they were killed by serum bactericidal activities more rapidly.

Magnitude of extended-spectrum β-lactamase and carbapenemase producing Enterobacteriaceae among commonly vended street foods in Arba Minch town, southern Ethiopia

BackgroundThe rising prevalence of extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae (ESβL-PE) in street foods poses a significant risk to human health due to its epidemiological significance. Thus, the aim of this study was to determine the magnitude of foodborne Enterobacteriaceae that produce carbapenemase and ESβL, as well as their patterns of antibiotic resistance, in the studied area.MethodsA community-based cross-sectional study was carried out from January 1st, 2023, to February 30th, 2023. One hundred randomly chosen street-vended food items (one hundred grams of each food item) were aseptically collected, and aliquots of 0.1 ml from the homogenized (25 g of samples into 225 ml of buffered peptone water (BPW)) were inoculated on MacConkey agar and Xylose Lysine Deoxycholate Agar (XLD). Enterobacteriaceae isolates were identified using various biochemical tests. ESβL and carbapenemase were first screened by indicator cephalosporins and carbapenem antibiotics, respectively. ESβL and carbapenemase were confirmed by a double-disc synergy test and modified carbapenem inactivation methods, respectively. Kirby-Bauer disc diffusion method was used for the antimicrobial-resistant test.ResultsA total of 112 Enterobacteriaceae belonging to six different genera were isolated. E. coli was attributed 39 (34.8%), followed by Citrobacter spp. 22 (19.6%) and K. pneumoniae 18 (16.1%), with only 8 (7.1%) isolated Salmonella spp. About 15.2% (n = 17) and 8.9% (n = 10) of Enterobacteriaceae were phenotypically confirmed to be extended-spectrum beta-lactamase (ESβL) and carbapenemase producers, respectively. The highest percentage of ESβL-producing isolates was attributed to K. pneumoniae (n = 5), E. coli (n = 4), and Enterobacter spp. (n = 3). Proteus spp. and Salmonella spp. isolates were carbapenemase-negative. All carbapenemase-positive isolates were found to be ESβL-producers. 70.6% (12/17) of ESβL-producing Enterobacteriaceae were found to be multidrug-resistant (MDR).ConclusionA considerable number of multidrug-resistant ESβL and carbapenemase-producing Enterobacteriaceae were identified, suggesting that street foods may be a potential source of MDR foodborne infections. Consequently, it is important to conduct routine examinations of street food items and track trends in medication resistance.

In-vitro Susceptibility Testing Methods for Ceftazidime-avibactam against Carbapenem-resistant Enterobacterales: Comparison with Reference Broth Microdilution Method.

β-lactam antibiotics, mainly cephalosporins, and carbapenems, have been the mainstay of treatment for infections caused by Enterobacterales. However, their role in treating clinical infections has become limited because of the increase in resistance. There is a need to have cost-effective and rapid methods for antimicrobial susceptibility testing methods for newer antibiotics like ceftazidime-avibactam against carbapenem-resistant Enterobacterales (CRE), which can be applied in routine clinical microbiology laboratories. With this aim, the present study was conducted to compare the disk diffusion and gradient diffusion, i.e., the E-test method with the reference broth microdilution (BMD) method for in-vitro testing of ceftazidime-avibactam against CRE. A total of 111 CRE isolates from various clinical samples were included. Conventional PCR (Polymerase Chain Reaction) was done for the detection of genes encoding carbapenemases and to see their expression, modified carbapenem inactivation method (mCIM) along with EDTA (Ethylenediaminetetraacetic acid) carbapenem inactivation method (eCIM) was done. 42.3% (47/111) isolates were resistant to ceftazidime-avibactam by the standard broth microdilution method; however, 45.9% (51/111) were resistant by both disk diffusion and E-test. In 5.4% of isolates (similar in both methods), microbroth dilution method results did not match with E-strip and disk diffusion. Very major errors (VME) by both disk diffusion and E-test were found in 2.1% (1/47), and major errors (ME) were found in 7.8% (5/64) isolates (similar isolates in both methods). The overall categorical agreement (CA) rate was 94.6% for both E-test and disk diffusion, and the essential agreement (EA) rate was 90.1% (100/111) for E-test. 98% (109/111) of CRE harbored carbapenemase genes either singly (30.3%) or in combination with others (69.7%). In conclusion, for CRE, E-test and the disk diffusion method for ceftazidimeavibactam depicted an acceptable performance as an alternative to the reference broth microdilution method.

Antibiotic resistance pattern of aerobic bacterial isolates from patients with skin and soft tissue infections in Karaikal

Skin and Soft Tissue Infections (SSTIs) are considered as non-fatal burden with significant morbidity and disability. The important challenge of severe SSTIs is choosing a drug for empirical treatment. From this region, only limited local antibiogram data is available. To determine the frequency of different aerobic bacteria isolated from patients with SSTIs attending dermatology and surgery departments of GH, Karaikal and to study the antibiotic resistance pattern of the isolates. This is a prospective, cross-sectional study with 100 samples. Standard protocol was followed for collection, processing, identification and antibiotic susceptibility testing. All isolates of and were screened for methicillin resistance and subsequently subjected to Oxacillin E-strip and Vancomycin E-strip to know the minimum inhibitory concentration (MIC) value. Isolates of Gram negative bacilli resistant to one or more carbapenems were tested for carbapenemase production using Modified Carbapenem Inactivation Method (MCIM) and multi drug resistant (MDR) organisms were identified. Most effective antibiotic for methicillin sensitive (MSSA) are Clindamycin (82.75%), Gentamicin (80.95%) and Cotrimoxazole (75%). The methicillin resistant (MRSA) incidence is 6.89% (2/29). Around 66.67% (4/6) of was Cefoxitin resistant. The carbapenem resistance was found to be 13.88% (5/36). Around 43.13% (22/51) Gram negative bacilli were MDR. The presence of MRSA and carbapenemase producing Gram negative bacilli are worrisome. Further, routine surveillance is needed to monitor the trends in antibiotic resistant pattern. However, this data paves way for judicious use of antibiotics for treatment and to prevent development of resistance in future.

Characterization of Transferrable Mechanisms of Quinolone Resistance (TMQR) among Quinolone-resistant Escherichia coli and Klebsiella pneumoniae causing Urinary Tract Infection in Nepalese Children

BackgroundTransferrable mechanisms of quinolone resistance (TMQR) can lead to fluoroquinolone non-susceptibility in addition to chromosomal mechanisms. Some evidence suggests that fluoroquinolone resistance is increasing among the pediatric population. We sought to determine the occurrence of TMQR genes among quinolone-resistant E. coli and K. pneumoniae causing urinary tract infections among Nepalese outpatient children (< 18 years) and identify molecular characteristics of TMQR-harboring isolates.MethodsWe performed antimicrobial susceptibility testing, phenotypic extended-spectrum β-lactamase (ESBL) and modified carbapenem inactivation method tests, and investigated the presence of six TMQR genes (qnrA, qnrB, qnrS, aac(6’)-Ib-cr, oqxAB, qepA), three ESBL genes (blaCTX−M, blaTEM, blaSHV), and five carbapenemase genes (blaNDM, blaOXA−48, blaKPC, blaIMP, blaVIM). The quinolone resistance-determining region (QRDR) of gyrA and parC were sequenced for 35 TMQR-positive isolates.ResultsA total of 74/147 (50.3%) isolates were TMQR positive by multiplex PCR [aac(6’)-Ib-cr in 48 (32.7%), qnrB in 23 (15.7%), qnrS in 18 (12.3%), qnrA in 1 (0.7%), and oqxAB in 1 (0.7%) isolate]. The median ciprofloxacin minimum inhibitory concentration of TMQR-positive isolates (64 µg/mL) was two-fold higher than those without TMQR (32 µg/mL) (p = 0.004). Ser-83→Leu and Asp-87→Asn in GyrA and Ser-80→Ile in ParC were the most common QRDR mutations (23 of 35). In addition, there was a statistically significant association between TMQR and two β-lactamase genes; blaCTX−M (p = 0.037) and blaTEM (p = 0.000).ConclusionThis study suggests a high prevalence of TMQR among the quinolone-resistant E. coli and K. pneumoniae isolates causing urinary tract infection in children in this area of Nepal and an association with the carriage of ESBL gene. This is a challenge for the management of urinary infections in children. Comprehensive prospective surveillance of antimicrobial resistance in these common pathogens will be necessary to devise strategies to mitigate the emergence of further resistance.

Evaluation of NG-Test CARBA 5 version 2, Cepheid Xpert Carba-R, and carbapenem inactivation methods in comparison to whole-genome sequencing for the identification of carbapenemases in non-fermenting Gram-negative bacilli.

NG-Test CARBA 5 (NG-Biotech) is a rapid in vitro multiplex immunoassay for the phenotypic detection and differentiation of the "big five" carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM). Version 2 of this assay was evaluated alongside the Xpert Carba-R assay (Cepheid, Inc.), the modified carbapenem inactivation method (mCIM), and the CIMTris assay, with a collection of carbapenem-resistant non-fermenting Gram-negative bacilli comprising 138 Pseudomonas aeruginosa and 97 Acinetobacter baumannii isolates. Whole-genome sequencing (WGS) was used as the reference standard. For P. aeruginosa, NG-Test CARBA 5 produced an overall percentage agreement (OPA) with WGS of 97.1%, compared with 92.8% forXpert Carba-R and 90.6% for mCIM. For A. baumannii, as OXA-type carbapenemases (non-OXA-48) are not included, both the NG-Test CARBA 5 and Xpert Carba-R only had an OPA of 6.2%, while the CIMTris performed well with an OPA of 99.0%. The majority of A. baumannii isolates (95.9%) tested falsely positive for IMP on NG-Test CARBA 5; no IMP genes were found on WGS. No clear cause was found for this phenomenon; a cross-reacting protein antigen unique to A. baumannii is a possible culprit. NG-Test CARBA 5 performed well for carbapenemase detection in P. aeruginosa. However, results from A. baumannii isolates should be interpreted with caution.

Fecal Carriage of Carbapenem Resistant Enterobacterales and Associated Factors Among Admitted Patients in Saint Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia.

The Enterobacterales family colonizes the human gut as normal flora in all age groups, with bacterial infections being the most common cause. Resistance is currently observed in all normal flora. The aim of this study was to determine the frequency of fecal carriage of carbapenem-resistant Enterobacterales (CRE), carbapenemase-producing Enterobacterales (CPE), and associated factors in the faeces of admitted patients. A cross-sectional study was conducted in Saint Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia. A total of 384 rectal swabs were collected from various wards in admitted patients who have consented to participate. The specimens were inoculated on a MacConkey agar plate, and then they were incubated at 37 °C for 18 to 24 hours. Using the BD PhoenixTM M50 compact system identification and antimicrobial susceptibility testing were performed. Using the modified carbapenem inactivation method, it was determined whether the carbapenem-resistant bacterial isolate produced carbapenemase or not. Overall prevalence of carbapenem-resistant Enterobacterales carriage and carbapenemase producing Enterobacterales in admitted patients was 17.2% (95%, Confidence Interval: 13.3-21.1%) and 7% (95%, Confidence Interval: 4.7-9.9%), respectively. The predominate carbapenem-resistant Enterobacterales in fecal carriage was K. pneumoniae, 15.4% (23/149), E. cloacae 15.4% (6/39), followed by E. coli 12.4% (37/307) of carbapenem-resistant Enterobacterales (CRE) isolate. Carbapenem-resistant Enterobacterales carriage isolates showed large level of resistance to ciprofloxacin, and sulfamethoxazole-trimethoprim. Prior intake of antibiotics (Odds Ratio 2.42, 95% CI: 11.186-4.95) was significantly associated with higher carbapenem-resistant Enterobacterales carriage. We observed a high prevalence of carbapenem-resistant Enterobacterales carriage and carbapenemase-producing Enterobacterales among admitted patients. There were only amikacin and colistin that could be effective for carbapenem-resistant Enterobacterales isolates. Hence, the control of carbapenem-resistant Enterobacterales carriage should be given priority by carbapenem-resistant Enterobacterales screening for fecal of admitted patients, and adhering to good infection prevention practice in hospital settings.

First Emergence of NDM-5 and OqxAB Efflux Pumps Among Multidrug-Resistant Klebsiella pneumoniae Isolated from Pediatric Patients in Assiut, Egypt.

New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae poses a high risk, especially among Egyptian pediatric patients who consume carbapenems antibiotics very widely and without adequate diagnostic sources. In addition, presence of efflux pump genes such as OqxAB increases resistance against many groups of antimicrobials which exacerbates the problem faced for human health. This study aimed to determine NDM variants among K. pneumoniae strains isolated from pediatric patients in Egypt, analyze the presence of OqxAB genes, and molecular characterization ofblaNDM-5-positiveK. pneumoniae. Fifty-six K. pneumoniaeisolates were recovered from pediatric patients, and tested for carbapenemase by modified carbapenem inactivation methods (mCIM) test. Minimum inhibitory concentrations of meropenem and colistin were determined by meropenem E-test strips and broth microdilution, respectively. PCR was used for the detection of the resistant genes (ESBL gene (blaCTX-M), carbapenemase genes (blaNDM, blaKPC) colistin resistant (mcr1, mcr2)) and genes for efflux pump (oqxA and oqxB). BlaNDM was sequenced. The effect of efflux pump in NDM-5-producing isolates was assessed by measuring MIC of ciprofloxacin and meropenem before and after exposure to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The horizontal gene transfer ability ofblaNDM-5 was determined using liquid mating assay and PCR-based replicon typing (PBRT) was done to determine the major plasmid incompatibility group. Twenty-nine isolates were positive for blaNDM-1, nine isolates were positive for blaNDM-5, and 15 isolates were positive for blaKPC. There is a significant increase of meropenem MIC of NDM-5-positive isolates compared with NDM-1-positive isolates. In addition, 38 isolates were positive for CTX-M, and 15 isolates were positive for mcr1. Both OqxA and OqxB were detected in 26 isolates and 13 isolates were positive for OqxA while 11 isolates were positive for OqxB only. All NDM-5-producing isolates except one isolate could transfer their plasmids by conjugation to their corresponding transconjugants (E. coli J53). Plasmid replicon typing showed that FII was predominant in NDM-5-producing K. pneumoniae. Similar strains were found between the three isolates and similarity was also detected between the two isolates. The highly resistant K. pneumoniae producing blaNDM-5 type was firstly isolated from pediatric patients. The association of efflux pump genes such as OqxAB is involved in resistance to ciprofloxacin. This highlighted the severity risk ofblaNDM-5-positiveK. pneumonia as it could transfer blaNDM-5 to other bacteria and has more resistance against carbapenems. This underlines the importance of continuous monitoring of infection control guidelines, and the urgent need for a national antimicrobial stewardship plan in Egyptian hospitals.

Isolation and Characterization of Carbapenem-Resistant Escherichia coli Carrying blaNDM and mcr-1 from Recurrent Urinary Tract Infection Patient.

The emergence of carbapenem-resistant E. coli (CRECO), leading to few antibacterial drugs available for CRECO infection. In this study, we report three carbapenem-resistant Escherichia coli (E. coli) isolates coproducing blaNDM and mcr-1 from patients with recurrent urinary tract infection (RUTI). Carbapenem-resistant E. coli strains, E55, E84, and E85, were isolated from the urine sample of RUTI patients. Antimicrobial susceptibility testing (AST) was conducted with VITEK-2 compact system and Kirby-Bauer (K-B) disk diffusion method. The ESBL test was detected by the disk diffusion method. The EDTA-modified carbapenem inactivation method (eCIM) and modified carbapenem inactivation method (mCIM) were performed for screening the carbapenemase. Multilocus sequence typing (MLST) was performed for molecular typing of the strains. The resistance genes were detected by PCR. The three isolates were all susceptible to tigecycline and nitrofurantoin. The blaNDM-1, blaCMY-6, blaTEM-1 and blaCTX-M-1, mcr-1, and porin loss expression of outer membrane protein F (OmpF) were detected in E55, which was assigned to ST2. The E84 and E85 were identified as ST471 carrying blaNDM-5, blaCTX-M55, and blaTEM-1 and the quinsolone-resistant genes aac(6')-Ib-cr and mcr-1. To our knowledge, our study is the first to report carbapenem-resistant E. coli strains carrying blaNDM and mcr-1 from urine of the recurrent urinary tract infection patients. These E. coli strains carrying blaNDM and mcr-1 should be closely monitored.

Complete-genome sequencing and comparative genomic characterization of blaNDM-5 carrying Citrobacter freundii isolates from a patient with multiple infections

BackgroundThe emergence and wide spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a growing threat to global public health. However, clinically derived carbapenemase-producing Citrobacter causing multiple infections has rarely been investigated. Here we first report the isolation and comparative genomics of two blaNDM-5 carrying Citrobacter freundii (C. freundii) isolates from a patient with bloodstream and urinary tract infections.ResultsAntimicrobial susceptibility testing showed that both blaNDM-5 carrying C. freundii isolates were multidrug-resistant. Positive modified carbapenem inactivation method (mCIM) and EDTA-carbapenem inactivation method (eCIM) results suggested metallo-carbapenemase production. PCR and sequencing confirmed that both metallo-carbapenemase producers were blaNDM-5 positive. Genotyping and comparative genomics analyses revealed that both isolates exhibited a high level of genetic similarity. Plasmid analysis confirmed that the blaNDM-5 resistance gene is located on IncX3 plasmid with a length of 46,161 bp, and could successfully be transferred to the recipient Escherichia coli EC600 strain. A conserved structure sequence (ISAba125-IS5-blaNDM-5-trpF-IS26-umuD-ISKox3) was found in the upstream and downstream of the blaNDM-5 gene.ConclusionsThe data presented in this study showed that the conjugative blaNDM-5 plasmid possesses a certain ability to horizontal transfer. The dissemination of NDM-5-producing C. freundii isolates should be of close concern in future clinical surveillance. To our knowledge, this is the first study to characterize C. freundii strains carrying the blaNDM-5 gene from one single patient with multiple infections.

DETECTION OF CARBAPENEMASE-PRODUCING CARBAPENEM-RESISTANT IN BLOOD CULTURE ISOLATES BY MCIM AND ECIM AND ITS SUSCEPTIBILITY TO TIGECYCLINE AND MINOCYCLINE

Objective: In this study, we aimed to detect different Carbapenemase-producing carbapenem-resistant Enterobacterales (CREs) in blood isolates by phenotypic, modified carbapenem inactivation methods (mCIM) and amp; EDTA-carbapenem inactivation methods (eCIM), and also to study the susceptibility of these CREs toward Tigecycline and Minocycline. Methods: This prospective study included 100 non-duplicate Enterobacterales organisms isolated from 250 blood samples positive for Enterobacterales that showed resistance to carbapenem (Imipenem). The isolates were identified by conventional routine biochemical tests. CRE isolates were screened for Carbapenemase production by the Clinical and Laboratory Standards Institute (CLSI)-recommended, mCIM and eCIM for evidence of the production of matello-beta-lactamase. Antimicrobial susceptibility for Tigecycline and Minocycline drugs was tested by the disk diffusion method on Mueller–Hinton agar according to CLSI guidelines, and susceptibility patterns were recorded. Clinical diagnosis data were collected from the requisition forms sent to our laboratory during test procedures. Results: Out of 100 (40%) CRE isolates tested for mCIM, 34 samples showed positive results for the Carbapenemase enzyme. Among the mCIM-positive isolates, Klebsiella spp. showed the highest prevalence of 58.8% (20/34). While among mCIM-positive isolates (22/34), 64.7% were positive for eCIM (Matello beta-lactamase producer). Maximum samples had been received from the NICU ward from patients diagnosed with early-onset sepsis; 41.2% of these were Carbapenemase-producing Enterobacterales. Among CRE isolates, 86 isolates were susceptible to Tigecycline and 24 isolates were susceptible to Minocycline. 70 CRE isolates were susceptible to Tigecycline but resistant to minocycline, and all CRE isolates resistant to Tigecycline were not susceptible to Minocycline.

Cefepime in vivo activity against carbapenem-resistant Enterobacterales that test as cefepime susceptible or susceptible-dose dependent in vitro: implications for clinical microbiology laboratory and clinicians.

Carbapenem-resistant Enterobacterales (CRE) are a public health concern. Among these isolates, there are reports of isolates that test as cefepime susceptible or susceptible-dose dependent (SDD) in vitro despite presence of a carbapenemase. This study aimed to evaluate the pharmacokinetic/pharmacodynamic profile of cefepime against carbapenemase-producing (CP-CRE) and non-producing (non-CP-CRE) isolates with a range of cefepime MICs. Reference broth microdilution and modified carbapenem inactivation method (mCIM) were performed on genotypically characterized clinical CRE isolates. Ultimately, CP-CRE (n = 21; blaKPC) and non-CP-CRE (n = 19) isolates with a distribution of cefepime MICs (≤0.5 to >256 mg/L) were utilized in the murine thigh infection model. Mice were treated with cefepime human-simulated regimens (HSRs) representative of a standard dose (1 g q12h 0.5 h infusion) or the SDD dose (2 g q8h 0.5 h infusion). Efficacy was assessed as the change in bacterial growth at 24 h compared with 0 h control, where ≥1 log bacterial reduction is considered translational value for clinical efficacy. Among both cohorts of CRE isolates, i.e. CP-CRE and non-CP-CRE, that tested as SDD to cefepime in vitro, 1 log bacterial reduction was not attainable with cefepime. Further blunting of cefepime efficacy was observed among CP-CRE isolates compared with non-CP-CRE across both susceptible and SDD categories. Data indicate to avoid cefepime for the treatment of serious infections caused by CRE isolates that test as cefepime susceptible or SDD. Data also provide evidence that isolates with the same antibiotic MIC may have different pharmacokinetic/pharmacodynamic profiles due to their antimicrobial resistance mechanism.

Carbapenem-resistant Klebsiella pneumoniae outbreak with monoclonal spread: Evaluation of resistance genes and ceftazidime-avibactam susceptibility

PurposeThe aim of this study was to investigate ceftazidime-avibactam (CAZ-AVI) susceptibility, carbapenemase genes, and clonal relationship in carbapenem-resistant Klebsiella pneumoniae (CrKp) isolates. MethodsA total of 28 non-repetitive CrKp isolates with positive carbapenemase production determined by the modified carbapenem inactivation method (mCIM), were included in the study. Identification of the isolates was performed with MALDI-TOF MS (VITEK-MS, bioMerieux, France). The automated system (VITEK-2, bioMerieux) and gradient diffusion test (Etest, bioMerieux) were used to determine antibiotic susceptibility. The mCIM was performed according to CLSI (2021) recommendations. CAZ-AVI susceptibility was carried out using the standard disc diffusion method. Results were evaluated according to EUCAST 2022 criteria. The blaOXA-48, blaNDM, blaKPC, blaIMP and blaVIM genes were investigated by multiplex PCR. The clonal relationship between isolates was determined by both AP-PCR and PFGE methods. ResultsOf the total 28 isolates, 89.3% were susceptible to CAZ-AVI. blaOXA-48 gene was found in 85.7% of the isolates, blaOXA-48+blaNDM gene in 10.7%, and blaNDM gene in 3.6%. blaKPC, blaIMP and blaVIM genes were not detected. Three clusters with three different genotypes were determined by the PFGE method. The largest cluster was Genotype A (n:24), followed by Genotype B (n:3), and Genotype C (n:1). AP-PCR was highly compatible with PFGE. The isolates of Genotype A, mostly from the intensive care unit (ICU), were evaluated as outbreak strains with monoclonal dissemination. ConclusionsOXA-48 remains the most frequently detected enzyme in CrKp strains in our country. The ceftazidime-avibactam susceptibility rate of 89.3% indicates that this antibiotic is still effective against CrKp isolates. The unnoticed outbreak detected in our study revealed the severity of intra-hospital cross-contamination affecting different wards, including the ICU. Therefore, in order to limit the spread of CrKp isolates, it is of great importance to implement strict infection control measures, and molecular surveillance programs, especially in the ICU.