Healthcare-associated infections pose a serious public health threat, as they are acquired during medical treatments or hospital stays, often leading to prolonged hospitalizations, high costs for healthcare systems and high mortality rates. Portugal has one of the highest rates of healthcare-associated infections in Europe, aggravated by the concerning rise in resistance to last-line antibiotics. These antibiotics are the last therapeutic alternative against infections by multidrug-resistant bacteria, generally used when other antibiotics are not effective. Bacteria of the genus Klebsiella are one of the main causes of these infections, and are usually treated with carbapenem antibiotics. However, bacteria that produce carbapenemases — enzymes that degrade these antibiotics — are on the rise, making treatment less and less effective.

Home / Publications / Publication

Home / Publications / Publication

DNA e Bactérias

THE RISING THREAT OF ANTIBIOTIC-RESISTANT KLEBSIELLA IN PORTUGUESE HOSPITALS

March 02, 25

Publication type: Article Summary
Original title: The role of mobile genetic elements in the dissemination of resistance genes in Klebsiella spp. clinical strains
Article publication date: November 2023
Source: Repositório Institucional do Instituto Superior Técnico (Scholar)
Author: João Francisco Ramalho
Supervisors: Cátia Caneiras & Leonilde Moreira

What is the goal, target audience, and areas of digital health it addresses?
     This study aims to provide insights into the landscape of resistance to a class of antibiotics called carbapenems in Klebsiella species present in Portuguese hospitals. The target audience includes microbiologists, infectious disease specialists, epidemiologists, hospital administrators responsible for implementing prevention strategies, and pharmaceutical companies involved in antibiotic development. In the context of digital health, the study contributes to epidemiological surveillance by using  bioinformatics tools and whole genome sequencing to identify resistance genes.

What is the context?
     Healthcare-associated infections pose a serious public health threat, as they are acquired during medical treatments or hospital stays, often leading to prolonged hospitalizations, high costs for healthcare systems and high mortality rates. Portugal has one of the highest rates of healthcare-associated infections in Europe, aggravated by the concerning rise in resistance to last-line antibiotics. These antibiotics are the last therapeutic alternative against infections by multidrug-resistant bacteria, generally used when other antibiotics are not effective. Bacteria of the genus Klebsiella are one of the main causes of these infections, and are usually treated with carbapenem antibiotics. However, bacteria that produce carbapenemases — enzymes that degrade these antibiotics — are on the rise, making treatment less and less effective.

     The genes encoding carbapenemase are mostly located in plasmids — small circular DNA molecules independent of the bacterial chromosome. These plasmids play a crucial role in antimicrobial resistance, as they can replicate independently and carry multiple resistance genes, allowing bacteria to rapidly adapt to antibiotics. In addition, they facilitate horizontal gene transfer, enabling resistance to spread across different environments and hosts, including humans and animals.  An example of this occurs when antibiotic-resistant bacteria are present in meat intended for human consumption. After ingestion, these bacteria can multiply in the gut and transfer their resistance genes to bacteria that already exist there.

What are the current approaches?
     Currently, the identification of antibiotic-resistant bacteria relies on a combination of laboratory techniques and bioinformatics tools.

     Traditional laboratory methods include culture-based susceptibility testing, such as the Kirby–Bauer disk diffusion technique, where an antibiotic impregnated disk is placed on a bacterial culture. After incubation, the presence of a clear zone around the disk indicates bacterial susceptibility, while its absence suggests resistance. For more precise quantification, the Epsilometer test (ETEST) is used to determine the minimum inhibitory concentration — the lowest antibiotic dose needed to inhibit bacterial growth — helping in choosing the appropriate dosage and assessing resistance to last-line antibiotics.

     Polymerase Chain Reaction (PCR) amplification is widely used to detect specific resistance genes. In this technique, researchers select a target gene and design primers — short DNA sequences — that define the points where amplification should begin and end. However, PCR only identifies previously known genes, limiting the detection of new mutations that confer resistance. To overcome this limitation, whole genome sequencing can be used, as it analyzes the entire bacterial genetic material, including chromosomes, plasmids, and other genetic elements. The DNA is fragmented, sequenced, and then reconstructed using bioinformatics tools, providing detailed insights into resistance genes, mutations, and genetic variations that drive antibiotic resistance.

     These techniques are further complemented by bioinformatics tools like BLAST (Basic Local Alignment Search Tool), which compares linear genetic sequences with a database to identify known resistance genes. The BLAST ring image generator, on the other hand, provides a circular map that visually compares a reference genome with multiple bacterial genomes at once, highlighting conserved genetic regions and unique resistance genes.

What does innovation consist of? How is the impact of this study assessed?
     This first large-scale genomic study of Klebsiella species in Portuguese hospitals combines advanced genetic, microbiological and bioinformatic analyses to uncover antibiotic resistance mechanisms and bacterial transmission. A key innovation was the discovery of a novel Klebsiella pneumoniae carbapenemase (KPC) gene, the blaKPC-98 gene, that compromises the effectiveness of last-resort antibiotic ceftazidime-avibactam. Submitted by the laboratory group, this gene was recognized as a new variant in GenBank.

     To contextualize the Portuguese findings at a global level, the study used GenBank, to identify carbapenemase genes and common Klebsiella strains worldwide.

     The study analyzed 1,140 clinical Klebsiella samples collected between 2019 and 2022 from three portuguese hospitals centers. Antibiotic multidrug resistance (resistance to three or more antibiotic classes) was assessed using the Kirby–Bauer disk diffusion method against six antibiotic classes. Next, the samples were submitted to PCR screening, followed by BLAST analysis to identify the Klebsiella strains and key carbapenemase genes (blaKPC, blaOXA-48, blaVIM, blaNDM, blaIMP, blaGES). From these, a diverse set of 218 samples were chosen to undergo whole genome sequencing and the obtained plasmids were aligned using BLAST ring image generator.

     The bioinformatics analysis included the use of the Kleborate tool to identify high-risk Klebsiella samples associated with hospital outbreaks, based on three genetic factors: (1) high-risk lineage defined by mutations in housekeeping genes (essential for survival), which are usually conserved but, when altered, they can acquire mutations that mark new lineages and allow transmission tracking, as these mutations are rarely lost; (2) presence of multidrug resistance genes; and (3) specific K-locus and O-locus genes, which encode surface sugar-based molecules that help bacteria evade detection by the host immune system.

     To characterize the blaKPC-98 gene, the study integrated genomic, microbiological, and bioinformatics analyses, comparing it to similar blaKPC genes to confirm its novelty. Gene cloning was performed to generate large quantities of bacteria carrying blaKPC-98, which were then subjected to susceptibility tests (Kirby–Bauer and ETEST) to verify resistance to ceftazidime-avibactam. In addition, the study also assessed the fitness cost, that is, the impact of the gene on bacterial survival. To evaluate this, blaKPC-98 gene was compared with blaKPC-3 gene through a long-term bacterial growth assay, culturing bacteria for 240 hours without antibiotics. The goal was to determine whether the novel resistance gene would be lost over time due to the expenditure of energy and resources required to produce the resistance protein. At the end of the experiment, bacteria carrying blaKPC-98 were again tested using the Kirby-Bauer method to verify whether they maintained resistance to ceftazidime-avibactam.

What are the main results? What are the main conclusions?
     Globally and in Portugal, Klebsiella pneumoniae is the predominant species within the genus Klebsiella (>93%). In Portugal, Klebsiella aerogenes is more common than Klebsiella quasipneumoniae, whereas globally, the trend is reversed. The distribution of carbapenemase genes varies by region: blaNDM-like genes dominates in Asia, blaKPC-3 and blaOXA-48 in Europe, and blaKPC-2 in America. In Portugal, blaKPC-3 is most prevalent, followed by blaOXA-181 and blaNDM-1. Only 20% of samples did not have carbapenemase genes, and several contained multiple carbapenemase genes, reinforcing the concern about the spread of resistance. The results of PCR and whole genome sequencing were concordant, confirming the robustness of the analysis.

     Most KPC-producing plasmids were large, carried multiple resistance genes, and were identified in various Klebsiella species, suggesting a highly conserved mechanism for horizontal gene transfer. Likewise, OXA-181-producing plasmids were also detected in different Klebsiella species, demonstrating the potential for dissemination of these genetic elements.

     All samples with the blaNDM-1 gene had the same plasmid (pNDM1_FMUL424), belonged to the same Klebsiella pneumoniae strain (ST11-KL105-O2), and originated from the same hospital (Hospital 3), confirming a hospital outbreak with possible transmission between patients. In contrast, Hospital 1 had other dominant Klebsiella pneumoniae lineages: ST17-KL25-O5, ST147-KL64-O2, ST13-KL3-O1, ST13-KL19-O1, and ST307-KL102-O2, while Hospital 2 was mainly affected by ST17-KL25-O5 and ST13-KL3-O1. The ST17-KL25-O5 and ST147-KL64-O2 lineages are globally widespread since the mid-1980s, whereas ST13 strains appear unique to Portuguese hospitals. A German outbreak was linked to ST307-KL102-O2, highlighting its potential for international spread.

     The study found that 98.9% of samples were multidrug-resistant, with high resistance rates to penicillins (99.1%), carbapenems (90%-97.4%), and cephalosporins (85.2%-96.5%). Meanwhile, ceftazidime-avibactam remained effective in most cases, with a relatively low resistance rate of 8.6% . This resistance profile underscores the urgency of monitoring emerging resistance mechanisms, such as the newly discovered blaKPC-98 gene, identified in the ST13-KL19-O1 lineage of Klebsiella pneumoniae. This gene has been confirmed to compromise the efficacy of ceftazidime-avibactam. However, the blaKPC-98 gene imposed a high fitness cost, leading to its loss after 240 hours without antibiotics exposure. In contrast, blaKPC-3 gene with a lower fitness cost, remained present. The tendency of bacteria to lose non-essential plasmids over generations allows them to optimize survival, but under sustained antibiotic pressure, plasmids carrying multiple resistance genes proliferate more efficiently, driving antimicrobial resistance in hospitals.

     To combat carbapenem-resistant Klebsiella in hospital settings, it is essential to strengthen epidemiological surveillance, adopt strict infection control measures and promote international collaboration through the sharing of data and good practices. Antibiotic stewardship programs should promote responsible antibiotic use, reducing selective pressure and preserving last-line treatments. Future research should include the analysis of environmental samples, such as hospital surfaces and water sources, to map transmission pathways and better understand bacterial resistance mechanisms.

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Home / Publications / Publication

DNA e Bactérias

THE RISING THREAT OF ANTIBIOTIC-RESISTANT KLEBSIELLA IN PORTUGUESE HOSPITALS

March 02, 25

Publication type: Article Summary
Original title: The role of mobile genetic elements in the dissemination of resistance genes in Klebsiella spp. clinical strains
Article publication date: November 2023
Source: Repositório Institucional do Instituto Superior Técnico (Scholar)
Author: João Francisco Ramalho
Supervisors: Cátia Caneiras & Leonilde Moreira

What is the goal, target audience, and areas of digital health it addresses?
     This study aims to provide insights into the landscape of resistance to a class of antibiotics called carbapenems in Klebsiella species present in Portuguese hospitals. The target audience includes microbiologists, infectious disease specialists, epidemiologists, hospital administrators responsible for implementing prevention strategies, and pharmaceutical companies involved in antibiotic development. In the context of digital health, the study contributes to epidemiological surveillance by using  bioinformatics tools and whole genome sequencing to identify resistance genes.

What is the context?
     Healthcare-associated infections pose a serious public health threat, as they are acquired during medical treatments or hospital stays, often leading to prolonged hospitalizations, high costs for healthcare systems and high mortality rates. Portugal has one of the highest rates of healthcare-associated infections in Europe, aggravated by the concerning rise in resistance to last-line antibiotics. These antibiotics are the last therapeutic alternative against infections by multidrug-resistant bacteria, generally used when other antibiotics are not effective. Bacteria of the genus Klebsiella are one of the main causes of these infections, and are usually treated with carbapenem antibiotics. However, bacteria that produce carbapenemases — enzymes that degrade these antibiotics — are on the rise, making treatment less and less effective.

     The genes encoding carbapenemase are mostly located in plasmids — small circular DNA molecules independent of the bacterial chromosome. These plasmids play a crucial role in antimicrobial resistance, as they can replicate independently and carry multiple resistance genes, allowing bacteria to rapidly adapt to antibiotics. In addition, they facilitate horizontal gene transfer, enabling resistance to spread across different environments and hosts, including humans and animals.  An example of this occurs when antibiotic-resistant bacteria are present in meat intended for human consumption. After ingestion, these bacteria can multiply in the gut and transfer their resistance genes to bacteria that already exist there.

What are the current approaches?
     Currently, the identification of antibiotic-resistant bacteria relies on a combination of laboratory techniques and bioinformatics tools.

     Traditional laboratory methods include culture-based susceptibility testing, such as the Kirby–Bauer disk diffusion technique, where an antibiotic impregnated disk is placed on a bacterial culture. After incubation, the presence of a clear zone around the disk indicates bacterial susceptibility, while its absence suggests resistance. For more precise quantification, the Epsilometer test (ETEST) is used to determine the minimum inhibitory concentration — the lowest antibiotic dose needed to inhibit bacterial growth — helping in choosing the appropriate dosage and assessing resistance to last-line antibiotics.

     Polymerase Chain Reaction (PCR) amplification is widely used to detect specific resistance genes. In this technique, researchers select a target gene and design primers — short DNA sequences — that define the points where amplification should begin and end. However, PCR only identifies previously known genes, limiting the detection of new mutations that confer resistance. To overcome this limitation, whole genome sequencing can be used, as it analyzes the entire bacterial genetic material, including chromosomes, plasmids, and other genetic elements. The DNA is fragmented, sequenced, and then reconstructed using bioinformatics tools, providing detailed insights into resistance genes, mutations, and genetic variations that drive antibiotic resistance.

     These techniques are further complemented by bioinformatics tools like BLAST (Basic Local Alignment Search Tool), which compares linear genetic sequences with a database to identify known resistance genes. The BLAST ring image generator, on the other hand, provides a circular map that visually compares a reference genome with multiple bacterial genomes at once, highlighting conserved genetic regions and unique resistance genes.

What does innovation consist of? How is the impact of this study assessed?
     This first large-scale genomic study of Klebsiella species in Portuguese hospitals combines advanced genetic, microbiological and bioinformatic analyses to uncover antibiotic resistance mechanisms and bacterial transmission. A key innovation was the discovery of a novel Klebsiella pneumoniae carbapenemase (KPC) gene, the blaKPC-98 gene, that compromises the effectiveness of last-resort antibiotic ceftazidime-avibactam. Submitted by the laboratory group, this gene was recognized as a new variant in GenBank.

     To contextualize the Portuguese findings at a global level, the study used GenBank, to identify carbapenemase genes and common Klebsiella strains worldwide.

     The study analyzed 1,140 clinical Klebsiella samples collected between 2019 and 2022 from three portuguese hospitals centers. Antibiotic multidrug resistance (resistance to three or more antibiotic classes) was assessed using the Kirby–Bauer disk diffusion method against six antibiotic classes. Next, the samples were submitted to PCR screening, followed by BLAST analysis to identify the Klebsiella strains and key carbapenemase genes (blaKPC, blaOXA-48, blaVIM, blaNDM, blaIMP, blaGES). From these, a diverse set of 218 samples were chosen to undergo whole genome sequencing and the obtained plasmids were aligned using BLAST ring image generator.

     The bioinformatics analysis included the use of the Kleborate tool to identify high-risk Klebsiella samples associated with hospital outbreaks, based on three genetic factors: (1) high-risk lineage defined by mutations in housekeeping genes (essential for survival), which are usually conserved but, when altered, they can acquire mutations that mark new lineages and allow transmission tracking, as these mutations are rarely lost; (2) presence of multidrug resistance genes; and (3) specific K-locus and O-locus genes, which encode surface sugar-based molecules that help bacteria evade detection by the host immune system.

     To characterize the blaKPC-98 gene, the study integrated genomic, microbiological, and bioinformatics analyses, comparing it to similar blaKPC genes to confirm its novelty. Gene cloning was performed to generate large quantities of bacteria carrying blaKPC-98, which were then subjected to susceptibility tests (Kirby–Bauer and ETEST) to verify resistance to ceftazidime-avibactam. In addition, the study also assessed the fitness cost, that is, the impact of the gene on bacterial survival. To evaluate this, blaKPC-98 gene was compared with blaKPC-3 gene through a long-term bacterial growth assay, culturing bacteria for 240 hours without antibiotics. The goal was to determine whether the novel resistance gene would be lost over time due to the expenditure of energy and resources required to produce the resistance protein. At the end of the experiment, bacteria carrying blaKPC-98 were again tested using the Kirby-Bauer method to verify whether they maintained resistance to ceftazidime-avibactam.

What are the main results? What are the main conclusions?
     Globally and in Portugal, Klebsiella pneumoniae is the predominant species within the genus Klebsiella (>93%). In Portugal, Klebsiella aerogenes is more common than Klebsiella quasipneumoniae, whereas globally, the trend is reversed. The distribution of carbapenemase genes varies by region: blaNDM-like genes dominates in Asia, blaKPC-3 and blaOXA-48 in Europe, and blaKPC-2 in America. In Portugal, blaKPC-3 is most prevalent, followed by blaOXA-181 and blaNDM-1. Only 20% of samples did not have carbapenemase genes, and several contained multiple carbapenemase genes, reinforcing the concern about the spread of resistance. The results of PCR and whole genome sequencing were concordant, confirming the robustness of the analysis.

     Most KPC-producing plasmids were large, carried multiple resistance genes, and were identified in various Klebsiella species, suggesting a highly conserved mechanism for horizontal gene transfer. Likewise, OXA-181-producing plasmids were also detected in different Klebsiella species, demonstrating the potential for dissemination of these genetic elements.

     All samples with the blaNDM-1 gene had the same plasmid (pNDM1_FMUL424), belonged to the same Klebsiella pneumoniae strain (ST11-KL105-O2), and originated from the same hospital (Hospital 3), confirming a hospital outbreak with possible transmission between patients. In contrast, Hospital 1 had other dominant Klebsiella pneumoniae lineages: ST17-KL25-O5, ST147-KL64-O2, ST13-KL3-O1, ST13-KL19-O1, and ST307-KL102-O2, while Hospital 2 was mainly affected by ST17-KL25-O5 and ST13-KL3-O1. The ST17-KL25-O5 and ST147-KL64-O2 lineages are globally widespread since the mid-1980s, whereas ST13 strains appear unique to Portuguese hospitals. A German outbreak was linked to ST307-KL102-O2, highlighting its potential for international spread.

     The study found that 98.9% of samples were multidrug-resistant, with high resistance rates to penicillins (99.1%), carbapenems (90%-97.4%), and cephalosporins (85.2%-96.5%). Meanwhile, ceftazidime-avibactam remained effective in most cases, with a relatively low resistance rate of 8.6% . This resistance profile underscores the urgency of monitoring emerging resistance mechanisms, such as the newly discovered blaKPC-98 gene, identified in the ST13-KL19-O1 lineage of Klebsiella pneumoniae. This gene has been confirmed to compromise the efficacy of ceftazidime-avibactam. However, the blaKPC-98 gene imposed a high fitness cost, leading to its loss after 240 hours without antibiotics exposure. In contrast, blaKPC-3 gene with a lower fitness cost, remained present. The tendency of bacteria to lose non-essential plasmids over generations allows them to optimize survival, but under sustained antibiotic pressure, plasmids carrying multiple resistance genes proliferate more efficiently, driving antimicrobial resistance in hospitals.

     To combat carbapenem-resistant Klebsiella in hospital settings, it is essential to strengthen epidemiological surveillance, adopt strict infection control measures and promote international collaboration through the sharing of data and good practices. Antibiotic stewardship programs should promote responsible antibiotic use, reducing selective pressure and preserving last-line treatments. Future research should include the analysis of environmental samples, such as hospital surfaces and water sources, to map transmission pathways and better understand bacterial resistance mechanisms.

Would you like to know all the details?

Know more here
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