Citrobacter spp. and Enterobacter spp. as reservoirs of carbapenemase blaNDM and blaKPC resistance genes in hospital wastewater.

Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gram-negative bacterial species. The presence of ß-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 ß-lactamase genes; blaTEM in 33.1%, blaCTX-M in 25.4%, blaKPC in 25.4%, blaNDM 8.8%, blaSHV in 5.3%, and blaOXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase blaKPC was found in six Citrobacter spp. and E. coli, while blaNDM was detected in two distinct Enterobacter spp. and E. coli. Notably, blaNDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase blaKPC and blaNDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains. IMPORTANCE: The significance of this study lies in its findings regarding the prevalence and diversity of antibiotic-resistant bacteria and genes identified in hospital wastewater in Mexico. The research underscores the urgent need for enhanced surveillance and prevention strategies to tackle the escalating challenge of antibiotic resistance, particularly evident through the elevated frequencies of carbapenemase genes such as blaKPC and blaNDM within the Enterobacteriaceae family. Moreover, the identification of these resistance genes on conjugative plasmids highlights the potential for widespread transmission via horizontal gene transfer. Understanding the mechanisms of antibiotic resistance in hospital wastewater is crucial for developing targeted interventions aimed at reducing transmission, thereby safeguarding public health and preserving the efficacy of antimicrobial therapies.

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.

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.

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.

Drug resistance phenotypes and genotypes in Mexico in representative gram-negative species: Results from the infivar network.

AIM: This report presents phenotypic and genetic data on the prevalence and characteristics of extended-spectrum ß-lactamases (ESBLs) and representative carbapenemases-producing Gram-negative species in Mexico. MATERIAL AND METHODS: A total of 52 centers participated, 43 hospital-based laboratories and 9 external laboratories. The distribution of antimicrobial resistance data for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa in selected clinical specimens from January 1 to March 31, 2020 was analyzed using the WHONET 5.6 platform. The following clinical isolates recovered from selected specimens were included: carbapenem-resistant Enterobacteriaceae, ESBL or carbapenem-resistant E. coli, and K. pneumoniae, carbapenem-resistant A. baumannii complex, and P. aeruginosa. Strains were genotyped to detect ESBL and/or carbapenemase-encoding genes. RESULTS: Among blood isolates, A. baumannii complex showed more than 68% resistance for all antibiotics tested, and among Enterobacteria, E. cloacae complex showed higher resistance to carbapenems. A. baumannii complex showed a higher resistance pattern for respiratory specimens, with only amikacin having a resistance lower than 70%. Among K. pneumoniae isolates, blaTEM, blaSHV, and blaCTX were detected in 68.79%, 72.3%, and 91.9% of isolates, respectively. Among E. coli isolates, blaTEM, blaSHV, and blaCTX were detected in 20.8%, 4.53%, and 85.7% isolates, respectively. For both species, the most frequent genotype was blaCTX-M-15. Among Enterobacteriaceae, the most frequently detected carbapenemase-encoding gene was blaNDM-1 (81.5%), followed by blaOXA-232 (14.8%) and blaoxa-181(7.4%), in A. baumannii was blaOXA-24 (76%) and in P. aeruginosa, was blaIMP (25.3%), followed by blaGES and blaVIM (13.1% each). CONCLUSION: Our study reports that NDM-1 is the most frequent carbapenemase-encoding gene in Mexico in Enterobacteriaceae with the circulation of the oxacillinase genes 181 and 232. KPC, in contrast to other countries in Latin America and the USA, is a rare occurrence. Additionally, a high circulation of ESBL blaCTX-M-15 exists in both E. coli and K. pneumoniae.

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.

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 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.

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.

Colonization by fecal extended-spectrum ß-lactamase-producing Enterobacteriaceae and surgical site infections in patients with cancer undergoing gastrointestinal and gynecologic surgery.

BACKGROUND: Cancer patients are at increased risk of infection. Fecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) may increase this risk. There are few studies on the prevalence of ESBL-PE colonization and surgical site infections (SSIs). METHODS: This prospective cohort study included patients with gastrointestinal and gynecological malignancies who were admitted to the hospital for elective surgery. Rectal swab cultures were obtained on the day of admission and during the postoperative period every 5 days. Prevalence of ESBL-PE fecal colonization and risk factors for the development of SSI were assessed. RESULTS: We included 171 patients, 30 (17.5%) of whom were colonized with ESBL-PE at admission. This proportion increased to 21% (37 of 171) of the samples during the hospital stay. Incidence of SSI was 14.6% (n = 25). Ten of 37 (27%) patients colonized by ESBL-PE developed SSI versus 15 of 134 (11%) of the non-ESBL-PE (relative risk [RR], 2.163; 95% confidence interval [CI], 1.201-3.897; P = .016). Five patients developed a bloodstream infection, and 4 patients were colonized with ESBL-PE (RR = 4.02; 95% CI, 1.2-3.89; P = .008). CONCLUSIONS: The rate of ESBL-PE fecal colonization in surgical patients was 17.5%. Colonization of ESBL-PE duplicated the risk of SSI by the same strain and, by a factor of 4, the risk of bloodstream infections.

Prevalence of the crpP gene conferring decreased ciprofloxacin susceptibility in enterobacterial clinical isolates from Mexican hospitals.

OBJECTIVES: This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. METHODS: A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. RESULTS: The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. CONCLUSIONS: These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.

Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans.

OBJECTIVE: Due to the fact that K. variicola, K. quasipneumoniae and K. pneumoniae are closely related bacterial species, misclassification can occur due to mistakes either in normal biochemical tests or during submission to public databases. The objective of this work was to identify K. variicola and K. quasipneumoniae genomes misclassified in GenBank database. MATERIALS AND METHODS: Both rpoB phylogenies and average nucleotide identity (ANI) were used to identify a significant number of misclassified Klebsiella spp. genomes. RESULTS: Here we report an update of K. variicola and K. Quasipneumoniae genomes correctly classified and a list of isolated genomes obtained from humans, plants, animals and insects, described originally as K. pneumoniae or K. variicola, but known now to be misclassified. CONCLUSIONS: This work contributes to recognize the extensive presence of K. variicola and K. quasipneumoniae isolates in diverse sites and samples.