Identification of Providencia spp. clinical isolates co-producing carbapenemases IMP-27, OXA-24, and OXA-58 in Mexico.

Providencia rettgeri, belonging to the genus Providencia, had gained significant interest due to its increasing prevalence as a common pathogen responsible for healthcare-associated infections in hospitals. P. rettgeri isolates producing carbapenemases have been reported to reduce the efficiency of carbapenems in clinical antimicrobial therapy. However, coexistence with other resistance determinants is rarely reported. The goal of this study was the molecular characterization of carbapenemase-producing Providencia spp. clinical isolates. Among 23 Providencia spp. resistant to imipenem, 21 were positive to blaNDM-1; one positive to blaNDM-1 and blaOXA-58 like; and one isolate co-producing blaIMP-27, blaOXA-24/40 like, and blaOXA-58 like were identified. We observed a low clonal relationship, and the incompatibility groups Col3M and ColRNAI were identified in the plasmid harboring blaNDM-1. We report for the first time a P. rettgeri strain co-producing blaIMP-27, blaOXA-24-like, and blaOXA-58 like. The analysis of these resistance mechanisms in carbapenemase co-producing clinical isolates reflects the increased resistance.

Molecular characterization of Escherichia coli producing extended-spectrum ß-lactamase CTX-M-14 and CTX-M-28 in Mexico.

The spread of ESBL-producing Escherichia coli has constantly increased in both clinical and community infections. Actually, the main ESBL reported is the CTX-M family, which is widely disseminated between the Enterobacteriaceae family. The epidemiology of the CTX-M family shows the CTX-M-15 variant dominating worldwide, followed by CTX-M-14 and CTX-M-27. The specific ESBL-producing E. coli clones included mainly the sequence types ST131, ST405, and ST648. In this report, we present the molecular characterization of ESBL-producing E. coli clinical isolates from eight hospitals in Mexico. From a collection of 66 isolates, 39 (59%) were identified as blaCTX-M-14 and blaCTX-M-27 belonging to the group CTX-M-9. We identified 25 (38%) isolates, producing blaCTX-M-28 belonging to the group CTX-M-1. blaCTX-M-2 and blaTEM-55 were identified in one isolate, respectively. Fourteen isolates (21%) were positive for blaCTX-M-14 (13%) and blaCTX-M-28 (7.3%) that were selected for further analyses; the antimicrobial susceptibility showed resistance to ampicillin (> 256 µg/mL), cefotaxime (> 256 µg/mL), cefepime (> 64 µg/mL), and ceftazidime (16 µg/mL). The ResFinder analysis showed the presence of the antimicrobial resistance genes aacA4, aadA5, aac(3)lla, sul1, dfrA17, tet(A), cmlA1, and blaTEM-1B. PlasmidFinder analysis identified in all the isolates the replicons IncFIB, which were confirmed by PCR replicon typing. The MLST analysis identified isolates belonging to ST131, ST167, ST405, and ST648. The ISEcp1B genetic element was found at 250 pb upstream of blaCTX-M-14 and flanked by the IS903 genetic element at 35 pb downstream. The IS1380-like element ISEc9 family transposase was identified at 250 pb upstream of blaCTX-M-14 and flanked downstream by the IS5/IS1182 at 80 pb. Our study highlights the significant prevalence of CTX-M-14 and CTX-M-28 enzymes as the second-most common ESBL-producing E. coli among isolates in Mexican hospitals. The identification of specific sequence types in different regions provides valuable insights into the correlation between ESBL and E. coli strains. This contribution to understanding their epidemiology and potential transmission routes is crucial for developing effective strategies to mitigate the spread of ESBL-producing E. coli in healthcare settings.

Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches / Los plásmidos del grupo de incompatibilidad FIBK se encuentran en Klebsiella variicola en diversos nichos ecológicos

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK (216/297) predominated in plasmids from human and environmental samples, followed by IncFIIK (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBF being most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.(AU)

Genetic characterization of plasmid-mediated fluoroquinolone efflux pump QepA among ESBL-producing Escherichia coli isolates in Mexico.

Antimicrobial resistance is a major global public health problem, with fluoroquinolone-resistant strains of Escherichia coli posing a significant threat. This study examines the genetic characterization of ESBL-producing E. coli isolates in Mexican hospitals, which are resistant to both cephalosporins and fluoroquinolones. A total of 23 ESBL-producing E. coli isolates were found to be positive for the qepA gene, which confers resistance to fluoroquinolones. These isolates exhibited drug resistance phenotypes and belonged to specific sequence types and phylogenetic groups. The genetic context of the qepA gene was identified in a novel genetic context flanked by IS26 sequences. Mating experiments showed the co-transfer of qepA1 and chrA determinants alongside blaCTX-M-15 genes, emphasizing the potential for these genetic structures to spread among Enterobacterales. The emergence of multidrug-resistant Gram-negative bacteria carrying these resistance genes is a significant clinical concern for public healthcare systems.

Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches.

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK (216/297) predominated in plasmids from human and environmental samples, followed by IncFIIK (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBF being most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.

Klebsiella pneumoniae K2 producer of pyogenic liver abscess associated with biliary communication.

INTRODUCTION: Hypervirulent strains of Klebsiella pneumoniae have gained clinical and epidemiological interest because of their capacity to cause severe and life-threatening infections. METHODOLOGY: We report a case involving infection with a hypervirulent K. pneumoniae K2 strain that caused liver abscess in a young woman with type 1 diabetes in Mexico. RESULTS: The infection was found to be associated with biliary tract communication. The virulence factors and capsular serotypes were identified by polymerase chain reaction analysis. After guided drainage and directed antibiotic treatment, the infection resolved and the patient recovered. Colonization of the gastrointestinal tract by hypervirulent K. pneumoniae strains, together with the presence of comorbidity, such as diabetes are important factors that contribute to the development of liver abscess. CONCLUSIONS: The identification of virulent clones is important to understand the pathogenicity and improve control of infections in the patients.

Molecular and Genomic Insights of mcr-1-Producing Escherichia coli Isolates from Piglets.

The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.

PCR system for the correct differentiation of the main bacterial species of the Klebsiella pneumoniae complex.

Accurate recognition of the closely related species Klebsiella pneumoniae, Klebsiella quasipneumoniae and Klebsiella variicola by phenotypic, biochemical and automated tests is notoriously unreliable in hospitals' diagnostic laboratories. A comparative genomics approach was conducted for the correct differentiation of the main bacterial species in the K. pneumoniae complex. Analysis of the deduced proteomes of 87 unique genomes of the Klebsiella in public databases, was used for the identification of unique protein family members. This allowed the design of a multiplex-PCR assay for the correct differentiation of these three species from different origins. This system allowed us to determine the prevalence of K. pneumoniae, K. quasipneumoniae and K. variicola among a collection of 552 clinical isolates. Of these, 87.3% (482/552) isolates corresponded to K. pneumoniae, 6.7% (33/552) to K. quasipneumoniae and 5.9% (33/552) to K. variicola. The multiplex-PCR results showed a 100% accuracy for the correct identification of the three species evaluated, which was validated with rpoB phylogenetic sequence analysis.

Exploring the environmental traits and applications of Klebsiella variicola.

Klebsiella variicola has been found in various natural niches, alone or in association with other bacteria, and causes diseases in animals and plants with important economic and environmental impacts. K. variicola has the capacity to fix nitrogen in the rhizosphere and soil; produces indole acetic acid, acetoin, and ammonia; and dissolves phosphorus and potassium, which play an important role in plant growth promotion and nutrition. Some members of K. variicola have properties such as halotolerance and alkalotolerance, conferring an evolutionary advantage. In the environmental protection, K. variicola can be used in the wastewater treatment, biodegradation, and bioremediation of polluted soil, either alone or in association with other organisms. In addition, it has the potential to carry out industrial processes in the food and pharmaceutical industries, like the production of maltose and glucose by the catalysis of debranching unmodified oligosaccharides by the pullulanase enzyme. Finally, this bacterium has the ability to transform chemical energy into electrical energy, such as a biocatalyst, which could be useful in the near future. These properties show that K. variicola should be considered an eco-friendly bacterium with hopeful technological promise. In this review, we explore the most significant aspects of K. variicola and highlight its potential applications in environmental and biotechnological processes.

Molecular characterization of KPC-2-producing Klebsiella pneumoniae ST258 isolated from bovine mastitis.

Bovine mastitis, an inflammation of the mammary gland of dairy cattle, is the most prevalent disease causing economically important losses, reduced milk production, early culling, veterinary expenses, and higher death rates. Bovine mastitis infections are the main cause for the use of antibiotics; however, the emergence of multidrug-resistant bacteria and the poor or nil response to antibiotics has become a critical global health problem. The goal of this study was the characterization of bacterial infections associated with clinical bovine mastitis. All the isolates were multidrug-resistant and were negative for the production of extended spectrum ß-lactamases. However, all isolates were identified as carbapenemase-producing organisms by the Carba NP test. The carbapenemase identified was the product of the KPC-2 gene. The isolates were identified as Klebsiella pneumoniae and contained virulence genes for fimbriae, lipopolysaccharides, nitrogen starvation genes, and siderophores. Sixty-nine percent of the KPC-2-producing isolates had the same plasmid profile, although the genetic mobilization of resistance by bacterial conjugation was unsuccessful. The carbapenemase corresponded to the plasmid-borne KPC-2 gene identified by Southern blot hybridization. The assay showed a positive signal in the 90 kb (69% of the isolates), 165 kb (31% of the isolates), and 130 kb (6% of the isolates) plasmids. The IncFIIy and IncFIIk replicons were detected among these K. pneumoniae isolates. The PFGE and MLST analysis showed that all of the isolates are comprised by two clones (A and B) belonging to Sequence Type 258. This is the first report of K. pneumoniae producing carbapenemase KPC-2 isolated from bovine mastitis.

ESBL-producing Escherichia coli and Klebsiella pneumoniae from health-care institutions in Mexico.

We investigated the phenotypic and molecular characteristics of Extended-Spectrum-ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates from four health-care institutions in Hermosillo, Sonora, Mexico. ESBL-producing isolates were collected from February to August 2016. The prevalence of ESBL-producing E. coli and K. pneumoniae was 11.9 and 8.7%, respectively. High dissemination of resistance to ciprofloxacin (88%), trimethoprim/sulfamethoxazole (72%) and aminoglycosides (59%) were detected, as well as susceptibility to meropenem, amikacin and tigecycline. The ESBL found variants were CTX-M-1 (88%) and CTX-M-9 (5%). The plasmid-mediated quinolone resistance (PMQR) gene aac(6´)-Ib-cr was identified in 62% of a representative sample, whereas the qnrB and qnrS genes were detected in 49% of the isolates. PFGE analyses detected many unrelated clones among the hospital or community isolates. A constant programme of epidemiological surveillance is recommended to understand the dynamics of bacterial resistance to both cephalosporin as well as the fluoroquinolone family of antibiotics.

Molecular epidemiology of Klebsiella variicola obtained from different sources.

Klebsiella variicola is considered an emerging pathogen in humans and has been described in different environments. K. variicola belongs to Klebsiella pneumoniae complex, which has expanded the taxonomic classification and hindered epidemiological and evolutionary studies. The present work describes the molecular epidemiology of K. variicola based on MultiLocus Sequence Typing (MLST) developed for this purpose. In total, 226 genomes obtained from public data bases and 28 isolates were evaluated, which were mainly obtained from humans, followed by plants, various animals, the environment and insects. A total 166 distinct sequence types (STs) were identified, with 39 STs comprising at least two isolates. The molecular epidemiology of K. variicola showed a global distribution for some STs was observed, and in some cases, isolates obtained from different sources belong to the same ST. Several examples of isolates corresponding to kingdom-crossing bacteria from plants to humans were identified, establishing this as a possible route of transmission. goeBURST analysis identified Clonal Complex 1 (CC1) as the clone with the greatest distribution. Whole-genome sequencing of K. variicola isolates revealed extended-spectrum ß-lactamase- and carbapenemase-producing strains with an increase in pathogenicity. MLST of K. variicola is a strong molecular epidemiological tool that allows following the evolution of this bacterial species obtained from different environments.

Klebsiella variicola: an emerging pathogen in humans.

The Klebsiella pneumoniae complex comprises seven K. pneumoniae-related species, including K. variicola. K. variicola is a versatile bacterium capable of colonizing different hosts such as plants, humans, insects and animals. Currently, K. variicola is gaining recognition as a cause of several human infections; nevertheless, its virulence profile is not fully characterized. The clinical significance of K. variicola infection is hidden by imprecise detection methods that underestimate its real prevalence; however, several methods have been developed to correctly identify this species. Recent studies of carbapenemase-producing and colistin-resistant strains demonstrate a potential reservoir of multidrug-resistant genes. This finding presents an imminent scenario for spreading antimicrobial resistant genes among close relatives and, more concerningly, in clinical and environmental settings. Since K. variicola was identified as a novel bacterial species, different research groups have contributed findings elucidating this pathogen; however, important details about its epidemiology, pathogenesis and ecology are still missing. This review highlights the most significant aspects of K. variicola, discussing its different phenotypes, mechanisms of resistance, and virulence traits, as well as the types of infections associated with this pathogen.

Molecular and genetic characterization of carbapenemase-producing bacteria in Venezuela.

Carbapenem-resistant Gram-negative bacteria isolated in Venezuela have been poorly characterized. The present study characterized a total of 34 isolates obtained from 27 patients; five of these patients were multi-infected. The bacterial species identified were Klebsiella pneumoniae (17), Pseudomonas aeruginosa (9), and Acinetobacter baumannii (8). From these isolates, 85% were identified as carbapenemase-producing bacteria, and the identified carbapenemase genes were blaKPC-2 (10/29 [34.4%]), blaVIM-type (7/29 [24.1%]), blaOXA-23 (7/29 [24.1%]), blaNDM-1 (8/29 [27.5%]), and the coexistence of blaOXA-23/blaNDM-1 (2/29 [6.8%]). Patient 1 was multi-infected by K. pneumoniae ST11 and ST2413 isolates harbouring the blaNDM-1 and blaKPC-2 genes, respectively. The other patients were multi-infected by two or three different bacterial species such as ESBL-producing K. pneumoniae isolates, P. aeruginosa harbouring the blaVIM-type gene, K. pneumoniae ST147 harbouring the blaKPC-2 gene and by A. baumannii harbouring the blaOXA-23 gene. The blaNDM-1 gene in A. baumannii is flanked by an uncommon genetic structure, whereas blaNDM-1 gene in K. pneumoniae revealed a common structure described in different plasmids from Enterobacteriaceae isolates. This study provides new information about the epidemiology of carbapenemase-producing bacteria in clinical setting in Venezuela.

Molecular characterization and pathogenicity determination of hypervirulent Klebsiella pneumoniae clinical isolates serotype K2 in Mexico.

Hypervirulent Klebsiella pneumoniae have been rarely described in Latin America. This work describes the characterization of hypervirulent K. pneumoniae isolates capsular serotype K2 belonging to sequence types 86 and 380. The assays showed the hypervirulent K. pneumoniae highly virulent, which is determined by the plasmid borne virulence genes. At this time, the hypervirulent K. pneumoniae clinical isolates in Mexico are extensively susceptible to antibiotics.

Cellular stress associated with the differentiation of Plasmodium berghei ookinetes.

For malaria transmission, Plasmodium parasites must develop in the mosquito vector. Oxidative stress in the insect midgut, triggered by environmental changes (e.g., pH and temperature), influences the cellular signaling involved in differentiation from gametocytes to mobile ookinetes for the purpose of parasite survival. Oxidative stress activates the homeostatic response to stress characterized by the phosphorylation eIF2α, the attenuation of protein synthesis, and the transcription of genes participating in the unfolded protein response and antioxidant processes, forming a part of an integrated stress response (ISR). We hypothesized that ISR operates during the differentiation of gametocytes to ookinetes to assure Plasmodium survival. Using in-vitro conditions resembling the mosquito midgut conditions, we cultured Plasmodium berghei gametocytes to ookinetes and evaluated the redox balance by detecting reactive oxygen species and superoxide dismutase activity. Additionally, we evaluated the phosphorylation of eIF2α, the attenuation of the global protein synthesis, and the gene expression of cellular stress markers (e.g., endoplasmic reticulum chaperones and antioxidant molecules, measured by reverse-transcription quantitative polymerase chain reaction), finding that these processes were all taking place, probably to improve survival during the differentiation of Plasmodium berghei ookinetes.

Sepsis, mitochondrial failure and multiple organ dysfunction.

PURPOSE: The purpose of this review is to consider the state of oxidative stress, failure of the antioxidant systems and mitochondrial failure as the main physiopathological mechanisms leading to multiple organ dysfunction during sepsis. PRINCIPAL FINDINGS: Sepsis is a clinical syndrome caused by a severe infection that triggers an exaggerated inflammatory response. Involved in the pathogenesis of sepsis are the activation of inflammatory, immune, hormonal, metabolic and bioenergetic responses. One of the pivotal factors in these processes is the increase of reactive species accompanied by the failure of the antioxidant systems, leading to a state of irreversible oxidative stress and mitochondrial failure. In a physiological state, reactive species and antioxidant systems are in redox balance. The loss of this balance during both chronic and infectious diseases leads to a state of oxidative stress, which is considered to be the greatest promoter of a systemic inflammatory response. The loss of the redox balance, together with a systemic inflammatory response during sepsis, can lead to progressive and irreversible mitochondrial failure, energy depletion, hypoxia, septic shock, severe sepsis, multiple organ dysfunction and death of the patient. CONCLUSION: Knowledge of the molecular processes associated with the development of oxidative stress should facilitate the development of effective therapies and better prognosis for patients with sepsis and organ dysfunction.