KPC Genes Research Articles

Molecular Characterization of a KPC-2- and NDM-1-Producing Klebsiella michiganensis Clinical Isolate in Cerebrospinal Fluid.

Klebsiella michiganensis is an emerging pathogen. In this context, we characterised a strain fxq isolated from a cerebrospinal fluid specimen of a patient with tentorial meningioma, and the K. michiganensis isolate produced carbapenemases of KPC and NDM types. The Phoenix 100 Automated Microbiology System, MALDI-TOF and whole-genome sequencing were used to identify the species. Anti-microbial susceptibility testing was also conducted with the Phoenix 100. The plasmid locations of the bla KPC-2 and bla NDM-1 genes were determined by S1-nuclease pulsed-field gel electrophoresis and Southern blot. The transfer capacity of plasmids carrying bla KPC-2 and bla NDM-1 was investigated by conjugation experiments, and the resistance plasmid stability was evaluated by culture and subculture. K. michiganensis subtypes were identified by multi-locus sequence typing. We performed whole-genome sequencing to confirm species, characterise plasmids and analyse core genes. fxq was originally identified as Klebsiella oxytoca and showed resistance to imipenem and meropenem, but whole-genome sequencing identified it to be K. michiganensis. The strain fxq belonged to the novel sequence type 202 (ST202) and carried the bla KPC-2 and bla NDM-1 genes located on the pB_KPC InFIA and pE_NDM IncU plasmids, respectively. The bla KPC-2-carrying plasmid was successfully transferred to Escherichia coli EC600 by conjugation, whereas the bla NDM-1 gene on the pE_NDM plasmid was not. The pB_KPC and pE_NDM plasmids demonstrated high stability. This work is the first report on a carbapenem-resistant clinical isolate K. michiganensis ST202 harbouring the bla KPC-2 and bla NDM-1 genes encoded by the IncFIA and IncU plasmids, respectively.

Phenotypic and genotypic characterization of extended spectrum beta-lactamase producing E. coli harboring carbapenem and colistin-resistant genes from poultry farms in Egypt.

eEscherichia coli (E. coli) bacteria that produce extended spectrum beta-lactamase (ESBL) is associated with a high prevalence of human illnesses worldwide. The emergence of resistance to carbapenem and colistin compounds poses further challenges to the treatment options for these illnesses. This study aimed to evaluate the phenotypic and genotypic pattern of resistance to carbapenem and colistin in ESBL-producing E. coli. Escherichia coli isolates collected from the respiratory tract of chickens in El-Sharkia government, Egypt. A total of 250 lung samples were collected from 50 poultry farms. These samples were then subjected to isolation, identification, and serotyping of E. coli. The presence of antimicrobial resistance was identified by disc diffusion testing. The occurrence of ESBL phenotypes was also assessed using the double disc synergy method. PCR/sequencing techniques were employed to examine the presence of ESBL (β-lactamase (bla)-TEM, blaSHV, and blaCTX-M), colistin (mcr-1), and carbapenem (blaNDM, blaVIM, and blaKPC) resistance genes. The findings revealed that 140 out of 250 (56%) were identified as E. coli. All E. coli isolates had a high level of multi-antimicrobial resistance (MAR) with an index value greater than 0.2, and 65.7% of them were confirmed to produce ESBL. Out of the 92 ESBL phenotypes, 55 (59.7%), 32 (34.7%), 18 (19.6%), and 37 (40.2%) isolates harbor b laTEM-3, b laSHV-4, b laCTX-M-1, a nd blaCTX-M-14 genes, respectively. The blaNDM-1 gene was identified in all 40 phenotypes that exhibited resistance to carbapenem, accounting for 28.5% of all strains of E. coli and 43.4% of ESBL isolates. The VIM and KPC genes were not detected in any of the samples. Furthermore, there was a significant prevalence of the mobilized colistin resistance (mcr)-1 gene, with 64 (69.5%) of the ESBL isolates exhibiting this gene. The prevalence of ESBL-producing E. coli, particularly those resistant to carbapenem and colistin, poses a significant public health risk in society.

Investigation of genotyping and phenotyping characteristics of carbapenem-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae (K. pneumoniae) is a major cause of healthcare-associated infections and plays a prominent role in the widespread antibiotic resistance crisis. Accurate identification of carbapenemases is essential to facilitate effective antibiotic treatment and reduce transmission of K. pneumoniae. This study aimed to detect carbapenemase production in carbapenem-resistant K. pneumoniae strains using phenotypic and genotypic methods. A total of 67 carbapenem-resistant K. pneumoniae strains obtained from various clinical samples were utilized for identification and antimicrobial susceptibility by the Vitek 2 Compact system (Biomerieux, France). Carbapenemase production was determined by using the Polymerase chain reaction, Blue-carba test (BCT) and Carbapenem inactivation method (CIM). Out of the isolates, 59 (88.1%) were positive bla OXA-48, 16 (23.9%) bla IMP, and five (7.5%) were positive bla NDM. No bla KPC genes were detected. The CIM identified 62 (92.5%), BCT identified 63 (94%) of PCR-positive isolates. The sensitivity and specificity of the BCT and the CIM were determined to be 96.7%, 40%, and 96.7%, 25% respectively. The bla OXA-48 gene was found to be the most prevalent in K. pneumoniae isolates. Early identification of carbapenem resistance plays a vital role in designing effective infection control strategies and mitigating the emergence and transmission of carbapenem resistance, thus reducing healthcare-associated infections.

The Klebsiella pneumoniae carbapenemase (KPC) β-Lactamase Has Evolved in Response to Ceftazidime Avibactam.

Klebsiella pneumoniae carbapenemase KPC is an important resistance gene that has disseminated globally in response to carbapenem use. It is now being implicated as a resistance determinant in Ceftazidime Avibactam (CAZ-AVI) resistance. Given that CAZ-AVI is a last-resort antibiotic, it is critical to understand how resistance to this drug is evolving. In particular, we were interested in determining the evolutionary response of KPC to CAZ-AVI consumption. Through phylogenetic reconstruction, we identified the variable sites under positive selection in the KPC gene that are correlated with Ceftazidime Avibactam (CAZ-AVI) resistance. Our approach was to use a phylogeny to identify multiple independent occurrences of mutations at variable sites and a literature review to correlate CAZ-AVI resistance with the mutations we identified. We found the following sites that are under positive selection: P104, W105, A120, R164, L169, A172, D179, V240, Y241, T243, Y264, and H274. The sites that correlate with CAZ-AVI resistance are R164, L169, A172, D179, V240, Y241, T243, and H274. Overall, we found that there is evidence of positive selection in KPC and that CAZ-AVI is the major selective pressure.

Multidrug Resistance and Molecular Characterization of Klebsiella spp. Isolated from the Cloacal Samples of Broiler Chickens in Bangladesh

Klebsiella spp. poses a significant zoonotic threat, capable of direct and indirect transmission from poultry farms to humans, resulting in severe conditions such as pneumonia, bloodstream infection, enteric fever, meningitis and urinary tract infections. The escalating use of antibiotics in the poultry industry is contributing to the rise of multidrug-resistant (MDR) microorganisms. Thus, a study was undertaken to comprehend the multidrug resistance and virulence gene profile of Klebsiella spp. isolated from poultry cloacal samples sourced from local markets of North Dhaka, Bangladesh. Following aseptic collection, the samples were transported to the laboratory for pure culture isolation. Employing standard microbiological methods such as mucoid colony characteristic, Gram-negative rod-shaped bacteria, biochemical tests and lactose fermentation in selective media, the isolated colonies were presumptively identified. Subsequently, an antibiotic susceptibility test, utilizing the disk diffusion assay, was conducted to assess drug sensitivity in these isolates. A PCR assay was performed to determine the presence or absence of KPC gene of Klebsiella. Among the isolates, ten colonies were identified as Klebsiella spp. based on the colony characteristics, Gram staining, biochemical tests and growth on selective media. Notably, eight isolates (excluding 2 and 4) were incapable of producing indole from tryptophan, indicating the presence of two different types of species. All isolates exhibited resistance to at least three antibiotics, namely ceftazidime, ampicillin and cefoxitin. Isolate 2 and 5 were demonstrated additional resistance to azithromycin, while isolate 9 and 10 exhibited further resistance to streptomycin and doxycycline. Molecular analysis indicated that 40% of these isolates harbor KPC virulent gene (880bp), with the most multidrug resistant isolate 9 and 10 possessing this gene. This study is pivotal for assessing the prevalence of virulent and MDR Klebsiella spp. in chicken cloacal samples, providing insights into shedding and transmission risk on soil and water environment, as well as to humans.

Detection of OXA-23 Gene in Amoxicillin-Clavulanic Acid-resistant Proteus mirabilis Isolates in Turkey

Class D carbapenemases, including OXA-23, OXA24/40, OXA-51, OXA-58, and OXA143, are typically associated with Acinetobacter species; OXA-48-like carbapenemases are usually found in Enterobacterales isolates. Although a few studies on Acinetobacter-type OXA genes in Enterobacterales have been reported, the presence of these Acinetobacter-type OXA genes in these isolates is quite limited. In contrast to other Enterobacterales isolates, studies have identified the presence of genes OXA-23, OXA-24, OXA-51, and OXA-58, which are typically associated with Acinetobacter species, Proteus mirabilis. This study aimed to investigate the presence of carbapenemase genes in amoxicillin-clavulanic acid-resistant P. mirabilis isolates. Thirty-two P. mirabilis isolates isolated from samples sent from intensive care patients and resistant to amoxicillin-clavulanic acid were included in the study. Vitek MS was used for bacterial identification, and the antibiotic resistance profile was determined by the Vitek 2 automated system. The presence of Acinetobacter type OXA genes (OXA-23, OXA-24, OXA-51, and OXA-58) and OXA-48, KPC, NDM, VIM, and IMP genes in P. mirabilis strains were investigated by polymerase chain reaction (PCR) method. In addition, ESBL production was detected in carbapenemase gene-positive isolates. According to the PCR results, while the presence of the OXA23 gene was detected in 15.6% of the isolates, the presence of OXA-24, OXA-51, OXA-58, OXA-48, KPC, NDM, VIM and IMP genes was not detected in any of the isolates. OXA-23, OXA-24, OXA-51, and OXA-58 are common carbapenemases in Acinetobacter species, especially OXA-23 is very common. However, these genes are very rare in Enterobacterales isolates. There are studies in which these genes were identified in P. mirabilis isolates. Similarly, in our research, the OXA-23 gene was detected in P. mirabilis isolates. To our knowledge, this is the first OXA-23 positivity reported in P. mirabilis isolates from our country.

Occurrence of Carbapenem-Resistant Enterobacteriaceae (Cre) Abhorring Bla Imp, Bla Kpc And Bla Oxa-48 Genes Recovered From Ready -To- Dispose Poultry Wastes In Osogbo, Osun State

The uncontrolled use of antimicrobials in animal husbandry such as poultry for prophylaxis reasons is to increase output for monetary gains which have led to several acquired community infections with high mortality and morbidity rates. This study is however aimed at investigating the occurrence, prevalence, and molecular detection of carbapenemase genes of carbapenem-resistant Enterobacteriaceae (CRE) bacteria isolated from poultry liters in Osogbo, Osun State Nigeria. Twenty ready-to-pack poultry waste samples were plated for the isolation of Enterobacteriaceae on MacConkey and Brilliance Escherichia coli agar. Disc diffusion method was employed for screening carbapenem resistant isolates while resistant genes were detected using carbapenem resistant gene primers. Forty-six Enterobacteriaeceae were isolated namely: Kluyvera ascorbate (28.3%), Klebsiella oxytoca (10.9%), Citrobacter koseri (8.7%), Klebsiella aereogenes, Raoultell (Klebsiella) planticola, Serratia nematodipila (6.5%), Trabulsiella laguamensis, Salmonella spp. (diarizonae), Proteus vulgaris, Citrobacter farmeri (4.3%), of which Edwardsiella tarda, Providencia rustigianii, Citrobacter freundii, Enterobacter cloacae, Cedecea lapagei, Serratia liquefaciens and Dickeyachry santhemi was (2.2%) respectively. 52.2% of the isolates were carbapenem resistant while 92% of the CRE had bla IMP genes. Only one CRE (Trabulsiella guamensis) was positive to bla KPC gene while four 16.6% of the isolates namely: Citrobacter koseri, Raoultell (Klebsiella) planticola, Serratia nematodipila, Citrobacter freundiu and Citrobacter koseri were positive to blaOXA-48 gene. Therefore, this study infers the high prevalence of CRE isolates in poultry litters as a public health concern for the indiscrimate use of carbapenem.
 Stamford Journal of Microbiology, 2023. Vol. 13, Issue 1, p. 15-20

Predominant transmission of KPC-2 carbapenemase in Germany by a unique IncN plasmid variant harboring a novel non-transposable element (NTE KPC -Y).

Current infection control protocols assume that the spread of KPC-2 carbapenemase-producing Enterobacterales (KPC2-CPE) by detected carriers to other in-house patients is through clonal transmission and can be restricted by implementing containment measures. We examined the presence of the bla KPC-2 gene in different genera and species of Enterobacterales isolated from humans at different hospitals and surface waters between 2013 and 2019 in Germany. We found that a single IncN[pMLST15] plasmid carrying the bla KPC-2 gene on a novel non-Tn4401-element (NTEKPC-Y), flanked by an adjacent region encoding 12 other antibiotic resistance genes, was uniquely present in multiple species of KPC2-CPE isolates. These findings demonstrate the selective impact of specific IncN plasmids as major drivers of carbapenemase dissemination and suggest "plasmid-based endemicity" for KPC2-CPE. Studies on the dynamics of plasmid-based KPC2-CPE transmission and its presence in persistent reservoirs need to be urgently considered to implement effective surveillance and prevention measures in healthcare institutions.

Investigation of the prevalence of carbapenem resistance genes in faecal carriage of carbapenem resistant Klebsiella spp. isolates by multiplex real-time PCR method.

Increased carbapenem resistance in Klebsiella spp. strains causes high morbidity and mortality. The genes encoded for carbapenemaseare transferrable between different bacterial species. In the present study, we aimed to investigate carbapenem resistance genes in Klebsiella spp. strains. Fifty Klebsiella spp. strains were isolated from rectal swabs of patients hospitalized in the neonatal intensive care unit (NICU). All strains were identified with API20E. The minimum inhibitory concentrations (MICs) of carbapenems were determined by the broth dilution method. The major five carbapenem genes (OXA-48, NDM, VIM, KPC, and IMP) were detected by the multiplex real-time PCR method. It was found that 49 (98%) of the strains were resistant to ertapenem (MIC ≥ 2μg/mL) and imipenem(MIC ≥ 4 μg/mL), and 47 (94%) of the strains were resistant to doripenem (MIC ≥ 4 μg/mL)and meropenem(MIC ≥ 4 μg/mL).NDM was detected in 42%, OXA-48 in 16%, and VIM in one (2%) isolate, and NDM + OXA-48 co-existed in 36% of the isolates. The KPC and IMP genes were not detected. NDM and NDM co-existing with OXA-48 were prevalent in the NICU of Istanbul Medical Faculty Hospital. Paying attention to the hand hygiene of healthcare workers, screening of rectal swabs of hospitalized patients for the presence of carbapenem resistance strains, and isolation of infected patients can effectively control the spread of carbapenem-resistant strains.

Evaluation of thymol effect on antimicrobial susceptibility and biofilm production in clinical Enterobacter spp. producing ESBLs and KPC enzymes

ABSTRACT Background Multidrug-resistant Enterobacter strains that harbour Klebsiella pneumoniaecarbapenemase and Extended Spectrum β-Lactamases genes are a therapeuticchallenge in hospitals that have made infection control and antibiotic therapydifficult and use of natural compounds such as thymol is interesting in thetreatment of infections. Methods Sixty-seven Enterobacter spp. isolates were obtained from patients admittedto hospitals in Hamadan and Tehran. Antimicrobial susceptibility, ESBLs andKPC genes detection and biofilm production of isolates were measured. Findings The results showed that the highest and lowest antibiotic resistance wererelated to ceftazidime (82.1%) and tigecycline (3%), respectively. Also, 79.1% of the isolates were MDR. The highestpresence of genes was related to TEM (100%), SHV (82%), CTX-M(77%) and KPC (8.96%) genes. The MICand MBIC of thymol were obtained 8-31 μg/ml and16-62 μg/ml, respectively. Conclusion Use of thymol, as an aid in antibiotic therapy is recommended.

The Significance of FilmArray Blood Culture Identification Panel (FA-BCID) for Managing Patients with Positive Blood Cultures.

We analyzed the accuracy and time efficiency of the FilmArray blood culture identification (FA-BCID) panel in identifying the pathogens in positive blood cultures. Two-hundred and seventy-two individuals were randomly assigned as the control (n = 212) and FA-BCID (n = 60) groups participating in this study. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to assess the control group. Meanwhile, the FA-BCID group was evaluated using both FA-BCID and MALDI-TOF, and the results were compared. The identification results from 73% (44/60) of the blood samples demonstrated agreement between FA-BCID and MALDI-TOF. The FA-BCID panel detected mecA genes in seven Staphylococcus species; six cases were confirmed using antimicrobial susceptibility testing. In addition, KPC genes were detected in one Escherichia coli and one Klebsiella pneumoniae, although only the latter corresponded with the result from antimicrobial susceptibility testing. The turnaround time (TAT) for identification through FA-BCID was shorter, with a median of 3.6 [2.4-4.6] hours (p < 0.05). No significant differences in the clinical and microbial outcomes following the ASP were observed between FA-BCID and MALDI-TOF. These results suggest that the FA-BCID panel provides an identification result that is as reliable as that provided by the routine identification procedure but with shorter TAT; thus, the FA-BCID method is considered an effective and beneficial method for therapeutic decision making and the improvement of the ASP for patients with bloodstream infection.

High prevalence of OXA-48-like and NDM carbapenemases among carbapenem resistant Klebsiella pneumoniae of clinical origin from Iran.

Klebsiella pneumoniae is increasingly developing resistance to last-resort antibiotics such as carbapenems. This study aimed to investigate the dissemination of common carbapenemase encoding genes among 48 clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP). Antimicrobial susceptibility testing was performed by broth dilution and disc diffusion methods. The phenotypic evaluation of carbapenemase production was performed by using Modified Carbapenem Inactivation Method. Presence of carbapenemase encoding genes blaKPC, blaNDM, blaOXA-48-like , blaIMP, and blaVIM was screened by PCR. Overall, carbapenemases were produced in all CRKP isolates. The blaOXA-48-like and blaNDM were the most prevalent genes detected among all and 66.6% (n=32) of CRKP isolates respectively. The blaVIM was detected in only one isolate co-harboring NDM and OXA-48-like carbapenemases. The blaKPC and blaIMP genes were not identified in any of the isolates. While tigecycline was the most active agent against CRKP isolates with low resistance rate (4.1%), high rate of resistance was observed to colistin (66.6%), amikacin (79%) and most of other tested antimicrobials. Our results revealed predominant prevalence of OXA-48-like and NDM carbapenemases among CRKP clinical isolates. High rate of resistance to last-resort agents such as colistin among CRKP isolates is a source of great concern.

B-066 Evaluation on Effectiveness of Colloidal Gold Immunochromatography Assay for Rapid Detection of Carbapenemase-producing Enterobacteriaceae

Abstract Background The identification, treatment, and control of carbapenem-resistant Enterobacteriaceae (CRE) infections are a major challenge for health care institutions and diagnostic laboratories worldwide. A novel Carbapenemase-producing Bacteria Test Kit (Lateral Flow Assay) from Dynamiker Biotechnology (Tianjin) Co., Ltd. is a qualitative test that was easy to operate and can be widely accepted by clinical and primary medical. Results can be read in just 15 minutes using colloidal gold immunochromatography, which can detect double and multiple carbapenemase types (Fig. 1). We analyzed the effectiveness of Dynamiker carbapenemase-producing bacteria test kit for rapid detection of CRE carbapenemase. Methods A total of 120 strains of Enterobacterales in blood culture were collected, including 80 strains of CRE and 40 strains of CSE (carbapenem-susceptibility Enterobacteriaceae). PCR was used to detect KPC, NDM, IMP, VIM and OXA-48 genes as gold standard. Dynamiker carbapenemase-producing bacteria test kit was used to detect the carbapenemase produced by CRE, which was conducted by the performance evaluation combined with PCR results. Results The 80 strains of CRE expressing carbapenemase genes were all positive for the detection of carbapenemasstre by Dynamiker carbapenemase-producing bacteria test kit, which includes 39 strains producing KPC, 26 strains producing NDM, 8 strains producing IMP, 5 strains producing VIM and 2 strains producing OXA-48 enzymes; the 40 strains of CSE were negative and no carbapenemase were detected. Compared with the PCR results, the sensitivity and specificity of the four enzymes by Dynamiker carbapenemase-producing bacteria test kit were both 100% (Fig. 2). Conclusion Dynamiker carbapenemase-producing bacteria test kit can be applied as a simple, rapid, sensitive and specific diagnostic method for the detection of different CRE carbapenemase types, which is of great value for the accurate use of clinical anti-infective therapy.Fig. 1.Operating procedures of colloidal gold immunochromatography.Fig. 2.Results of Dynamiker carbapenemase-producing bacteria test kit C: Control, K: KPC, N: NMD, I: IMP, V: VIM, O: OXA-48.

Molecular characterization of Carbapenem-resistant Escherichia coli isolates from sewage at Mulago National Referral Hospital, Kampala: a cross-sectional study

BackgroundEscherichia coli (E. coli) is one of the most frequent causes of fatal bacterial infections affecting both humans and animals. The resistance to Carbapenems is mainly associated with enzyme-mediated resistance mechanism, through the acquisition of Carbapenemase genes. In Uganda, no studies have been done to detect presence of Carbapenem-resistant E. coli in sewage. We therefore carried out a study to characterize Carbapenem-resistant E. coli from sewage from Mulago National Referral Hospital.Methods and resultsIn this cross-sectional study, a total of 104, sewage samples were aseptically collected, cultured on MacConkey agar supplemented with Meropenem 1 µg/ml with other standard microbiology methods to screen for Carbapenem-resistant E. coli (CREC). Antimicrobial susceptibility testing was performed on the CREC, using Imipenem (10 mg/disc) and Meropenem (10 mg/disc), Carbapenem drugs readily available on market. Multiplex PCR was performed on selected Carbapenem-resistant and susceptible isolates to detect Carbapenemase genes. Later the isolates were pathotyped for virulence genes that included pathogenicity islands (PAIs) and phylogenetic markers. The results showed that the Carbapenem-resistant E. coli isolates were more resistant to Meropenem (64%) than Imipenem (60%). KPC gene was the most predominant (75%), followed by NDM gene (30%) while no OXA-48, IMP-1, and IMP-2 genes were detected. Pathotyping of virulence genes showed presence of eae gene, as the most predominant (40%), followed by elt gene (25%) and negative for stx and aggR genes. For PAI markers, only the PAI IV536 gene was detected at 10%. Then, pathotyping of the phylogenetic markers was present in 85% of the typed isolates with yjaA gene the most abundant (60%) while both chuA and TSPE4.C2 were detected in 5% of the isolates.ConclusionBoth pathogenic and non-pathogenic Carbapenem-resistant E. coli strains are present in the sewage of Mulago National Referral Hospital in Uganda.

Endogenous Bacteremia Caused by Intestinal Colonization of Carbapenem-Resistant Enterobacteriaceae (CRE) in Immunocompromised Children.

Carbapenem-resistant Enterobacteriaceae (CRE) infection is life-threatening, especially for immunocompromised children. The source tracking of CRE could prevent bacteremia during hospitalization. In this study, the intestinal colonization of CRE and their translocation to blood were investigated. Stool samples from immunocompromised pediatric patients were collected after admission, and secondary stool and blood samples were collected in case of fever. After CRE phonotypic detection, the OXA-48, NDM-1, VIM, IMP, and KPC genes were detected by PCR. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) was used to determine the phylogenic relatedness of the blood and fecal isolates. Bacteremia was recorded in 71.4% of the patients. Enterobacteriaceae spp. were recorded in 100% of the stool samples and 31% of the blood samples. The correlation between the length of stay (LOS), days of fever, chemotherapy regimens, and death rate was significant (p-value ≤ 0.05). OXA-48 was present in all CRE isolates in both the primary and the secondary stool samples and the blood samples. According to the phylogenetic data, 58.33% of the patients with bacteremia had identical blood and stool isolates. The death rate was 24.4% in children with CRE bacteremia. The primary intestinal colonization with CRE in immunocompromised pediatrics and their translocation to blood was established in this study. The implementation of infection control programs and the application of infection prevention strategies for immunocompromised children is necessary.

Simultaneous and Visual Detection of KPC and NDM Carbapenemase-Encoding Genes Using Asymmetric PCR and Multiplex Lateral Flow Strip.

Carbapenem-resistant Enterobacteriaceae (CRE) infections constitute a threat to public health, and KPC and NDM are the major carbapenemases of concern. Rapid diagnostic tests are highly desirable in point-of-care (POC) and emergency laboratories with limited resources. Here, we developed a multiplex lateral flow assay based on asymmetric PCR and barcode capture probes for the simultaneous detection of KPC-2 and NDM-1. Biotinylated barcode capture probes corresponding to the KPC-2 and NDM-1 genes were designed and cast onto two different sensing zones of a nitrocellulose membrane after reacting with streptavidin to prepare a multiplex lateral flow strip. Streptavidin-coated gold nanoparticles (SA-AuNPs) were used as signal reporters. In response to the target carbapenemase genes, biotin-labelled ssDNA libraries were produced by asymmetric PCR, which bond to SA-AuNPs via biotin and hybridise with the barcode capture probe via a complementary sequence, thereby bridging SA-AuNPs and the barcode capture probe to form visible red lines on the detection zones. The signal intensities were proportional to the number of resistance genes tested. The strip sensor showed detection limits of 0.03 pM for the KPC-2 and 0.07 pM for NDM-1 genes, respectively, and could accurately distinguish between KPC-2 and NDM-1 genes in CRE strains. For the genotyping of clinical isolates, our strip exhibited excellent consistency with real-time fluorescent quantitative PCR and gene sequencing. Given its simplicity, cost-effectiveness, and rapid analysis accomplished by the naked eye, the multiplex strip is promising auxiliary diagnostic tool for KPC-2 and NDM-1 producers in routine clinical laboratories.

The Presence of Virulent and Multidrug-Resistant Clones of Carbapenem-Resistant Klebsiella pneumoniae in Southeastern Brazil.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) represents an urgent threat to global public health due to the limited therapeutic options available to control this pathogen. This study aims to analyze the molecular epidemiology, antimicrobial resistance and virulence profile of CRKP isolated from patients at hospitals in Southeastern Brazil. KPC and other beta-lactamase genes were detected in all strains, which were also multidrug-resistant (MDR). In addition, 11 strains showed resistance to last-resort antimicrobials, such as colistin and tigecycline. MLST analysis revealed eight different sequence types (ST11, ST37, ST147, ST340, ST384, ST394, ST437, and ST628), being two (ST628 and ST394) reported for the first time in Brazil. Strains belonging to the clonal complex 258 (CC258) "high-risk clones" were prevalent in this study. The Galleria mellonella model showed the emergence of virulent CRKP strains in the healthcare environment and, suggests that colistin-resistant strains were associated with higher virulence. This study shows the presence of virulent CRKP-MDR strains in hospitals across Southeastern Brazil, and draws attention to the presence of highly virulent emerging CRKP-MDR ST628 strains, showing that virulent and resistant clones can emerge quickly, requiring constant monitoring.

Phenotypic and Genotypic Detection of Carbapenem Resistant Organism Causing Nosocomial Infection in Patients Attending Tertiary Care Hospital

Aims: Nosocomial infections are the leading cause of mortality. ESKAPE organisms are the primary causes of nosocomial infection as these organisms are more or less carbapenem resistant. This study aimed to isolate and identify the etiological agents responsible for causing nosocomial infection and determine the carbapenemase producing organism by phenotypic and genotypic detection.&#x0D; Study Design: The study design is cross-sectional study.&#x0D; Place and Duration of Study: The study was conducted in the Department of Microbiology, at Index Medical College, Indore, between January 2020 and January 2022.&#x0D; Methodology: Total of 246 samples was collected from the patients who develop symptoms after 48- 72 hrs of hospitalization. Samples were processed for identification of etiological agents. Gram negative organisms were selected and further identified for carbapenemase enzyme. Screening was done by Kirby Bauer disc diffusion test and further confirmed by Modified Hodge test, Combined Disc test, Double Disc Synergy Diffusion test and Carbapenem Inactivation method. Genotypic detection was done by using multiplex polymerase chain reaction for KPC, NDM and OXA-48 gene.&#x0D; Results: Out of 107-gram-negative organisms, 19 (17.75%) were carbapenem resistant. Among 19 carbapenem resistant GNR, 13% MHT, 15% CCDT, 17% DDST and 17% mCIM were positive. The sensitivity and specificity of MHT, CCDT, DDST, mCIM were 74%/100, 84%/100,95%/100 and 95%/100respectively. The genotypic detection shows highest percentage of blaNDM 74% which is followed by bla OXA-48 31% and blaKPC 26%.&#x0D; Conclusion: Hospitals have become the hotspot for various microorganism causing nosocomial infections and are getting carbapenem resistance due to irrational use of antibiotics. Antimicrobial stewardship is one of the effective measures that minimize the resistance. Proper universal precaution can also minimize, spread of resistance in organism. If the last resort drug gets resistant, then it could be challenging for the clinician to treat their patients. Hospitals should have regular HAI meeting and release of antibiogram to know the pattern of these notorious organisms invading infection.

Prevalence of Carbapenem-Resistant Enterobacteriaceae and the Genes Responsible for Carbapenemase Production in a Tertiary Care Hospital in South India

Introduction: Carbapenem resistance in Gram-negative bacilli (GNB) is a major concern in the management of resistant infections. The mechanism of carbapenem resistance is most commonly mediated by carbapenemases. The five most common genes (NDM, KPC, VIM, OXA, and IMP) are responsible for carbapenemase production. Knowledge of these genes is important for the management of the disease. Objective: To estimate the prevalence of different genes responsible for carbapenemase production in GNB at a tertiary healthcare centre in South India. Method: In this retrospective study, samples were collected over 16 months. Carbapenem-resistant GNB underwent to Xpert Carba-R assay (Cepheid, Sunnyvale, California, USA) for the detection of five important genes responsible for carbapenemase production: NDM, KPC, VIM, OXA, and IMP. Results: Out of 184 carbapenem-resistant GNB, 20 samples were not included in this study. The rest of the 164 samples grew Klebsiella pneumoniae (152), Escherichia coli (10), and Enterobacter (2). OXA-48 and NDM were the most common genes responsible, with 137 (84.5%) and 95 (58.6%), respectively. Among them, 70 (43.2%) showed the presence of both genes, and 1 (0.6%) showed the presence of OXA-48, NDM, and VIM. Individually, 66 (40.7%) of OXA-48, 24 (14.8%) of NDM, and one (0.6%) of VIM. In this study, the authors did not find the presence of IMP or KPC genes. Conclusion: As a result of limited options and the higher cost of antibiotics for carbapenem-resistant infections, knowledge of these genes helps in the selection and rational use of antibiotics reduces the cost of management and will prevent mortality and morbidity from these infections.

Epidemiological and Molecular Characteristics of bla NDM-1 and bla KPC-2 Co-Occurrence Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged and spread worldwide. It can usually cause a serious threat complicating treatment options in clinical settings. However, treatment options are limited. The present study investigates the prevalence and genetic characteristics ofbla NDM-1 and bla KPC-2co-harboring clinical isolates ofKlebsiella pneumoniae. In this study, Multiplex polymerase chain reaction (PCR) was performed to detect the carbapenem-resistant genes, and the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of antibacterial drugs. The transferability of carbapenem-resistant phenotypes was examined using filter mating assays. Overall, we used Illumina sequencing to evaluate the epidemiological and molecular characteristics of bla NDM-1 and bla KPC-2 (genes encoding carbapenemase) co-occurrence in CRKP strains. All strains exhibited resistance to carbapenems and other antibiotics. However, they were still susceptible to polymyxin E. Among them, 18 isolates were positive for bla KPC-2, bla NDM-1, and multiple virulence determinants, such as genes encoding the virulence factor aerobactin, yersiniabactin, and the regulator of the mucoid phenotype (rmpA and rmpA2). Whole genome sequencing revealed that the 18 CRKP strains belonged to ST11 and capsular serotype KL64, and could be grouped into two evolutionary branches. Furthermore, these strains displayed hypervirulence potential since all of them carried pLVPK-like plasmid. These findings suggested that ST11-KL64 CRKP strains are major threats in terms of nosocomial infections in this hospital. Hence, new strategies should be urgently developed to monitor, diagnose, and treat this high-risk CRKP clone.