Relevance of the Adjuvant Effect between Cellular Homeostasis and Resistance to Antibiotics in Gram-Negative Bacteria with Pathogenic Capacity: A Study of Klebsiella pneumoniae.

Antibiotic resistance has become a global issue. The most significant risk is the acquisition of these mechanisms by pathogenic bacteria, which can have a severe clinical impact and pose a public health risk. This problem assumes that bacterial fitness is a constant phenomenon and should be approached from an evolutionary perspective to develop the most appropriate and effective strategies to contain the emergence of strains with pathogenic potential. Resistance mechanisms can be understood as adaptive processes to stressful conditions. This review examines the relevance of homeostatic regulatory mechanisms in antimicrobial resistance mechanisms. We focus on the interactions in the cellular physiology of pathogenic bacteria, particularly Gram-negative bacteria, and specifically Klebsiella pneumoniae. From a clinical research perspective, understanding these interactions is crucial for comprehensively understanding the phenomenon of resistance and developing more effective drugs and treatments to limit or attenuate bacterial sepsis, since the most conserved adjuvant phenomena in bacterial physiology has turned out to be more optimized and, therefore, more susceptible to alterations due to pharmacological action.

Pyogenic liver abscess caused by an atypical hypervirulent Klebsiella pneumoniae K1-ST23 in Mexico.

Hypervirulent K. pneumoniae infection has been raising worldwide and is one of the major causes of community-acquired pyogenic liver abscess. We described a case report of pyogenic liver abscess caused by an atypical hypervirulent (non-hypermucoviscous) K. pneumoniae K1 ST23 in a diabetic Asian patient who resided in Mexico. The susceptibility to antimicrobials, pathogenicity, molecular and genomic analysis were determined. A man from Guangdong (China) with a recent diagnosis of diabetes mellitus was admitted to the hospital, and he denied traveling in the last 3 months. A computed tomography revealed a right lobe liver abscess. On the third day after admission a Klebsiella pneumoniae isolate (14652) was obtained. The isolate corresponded to a susceptible K. pneumoniae with capsular type K1 and ST23 (CG23) and exhibited a non-hypermucoviscous phenotype. The isolate 14652 was genetically related to the globally distributed lineage ST23-KL1. This study describes the first case in Mexico of K. pneumoniae capsular type K1 and ST23 with an atypical hypervirulent phenotype.

Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates.

Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-ß-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

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.

Genome sequence of a virulent and hypermucoviscous-like Klebsiella michiganensis clinical isolate.

OBJECTIVES: The hypermucoviscous-like phenotype has been described in Klebsiella pneumoniae species complex (KpSC) and was described as a contributor of increased virulence. This study described the characterization and whole-genome sequencing of an antibiotic susceptible and hypermucoviscous-like Klebsiella michiganensis 9273 clinical isolate. DATA DESCRIPTION: Here, we report the genome sequence of a K. michiganensis clinical isolate obtained from a urinary tract infection exhibiting the hypermucoviscous-like phenotype. The draft genome sequence consisted of 145 contigs and ~ 6.6 Mb genome size. The annotation revealed 6648 coding DNA sequences and 56 tRNA genes. The strain belongs to the sequence type (ST) 50, and the OXY-1 beta-lactam resistance gene, aph(3')-Ia gene for aminoglycoside resistance and multidrug efflux pumps were identified. The fyuA siderophore receptor of yersiniabactin siderophore was identified. Increased virulence was observed in Galleria mellonella larvae model and increased capsule production was determined by uronic acid quantification. The clinical implications of this phenotype are unknown, but the patient outcome might worsen compared to susceptible- or MDR-classical K. michiganensis isolates.

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.

Genome Sequences of Bordetella pertussis Isolates from Outbreaks in Northeastern Mexico.

Here, we report the draft genome sequences of 4 Bordetella pertussis isolates which correspond to major clones isolated between 2008 and 2014 from two outbreaks in northeastern Mexico. The B. pertussis clinical isolates belong to the ptxP3 lineage, and they are grouped into two major clusters, defined by the fimH allele.

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.

Whole genome analysis of Gram-negative bacteria using the EPISEQ CS application and other bioinformatic platforms.

OBJECTIVES: To determine genomic characteristics and molecular epidemiology of carbapenem non-susceptible Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa from medical centres of Mexico using whole genome sequencing data analysed with the EPISEQⓇ CS application and other bioinformatic platforms. METHODS: Clinical isolates collected from 28 centres in Mexico included carbapenem-non-susceptible K. pneumoniae (n = 22), E. coli (n = 24), A. baumannii (n = 16), and P. aeruginosa (n = 13). Isolates were subjected to whole genome sequencing using the Illumina (MiSeq) platform. FASTQ files were uploaded to the EPISEQⓇ CS application for analysis. Additionally, the tools Kleborate v2.0.4 and Pathogenwatch were used as comparators for Klebsiella genomes, and the bacterial whole genome sequence typing database was used for E. coli and A. baumannii. RESULTS: For K. pneumoniae, both bioinformatic approaches detected multiple genes encoding aminoglycoside, quinolone, and phenicol resistance, and the presence of blaNDM-1 explained carbapenem non-susceptibility in 18 strains and blaKPC-3 in four strains. Regarding E. coli, both EPISEQⓇ CS and bacterial whole genome sequence typing database analyses detected multiple virulence and resistance genes: 20 of 24 (83.3%) strains carried blaNDM, 3 of 24 (12.4%) carried blaOXA-232, and 1 carried blaOXA-181. Genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, phenicols, trimethoprim, and macrolides were also detected by both platforms. Regarding A. baumannii, the most frequent carbapenemase-encoding gene detected by both platforms was blaOXA-72, followed by blaOXA-66. Both approaches detected similar genes for aminoglycosides, carbapenems, tetracyclines, phenicols, and sulfonamides. Regarding P. aeruginosa, blaVIM, blaIMP, and blaGES were the more frequently detected. Multiple virulence genes were detected in all strains. CONCLUSION: Compared to the other available platforms, EPISEQⓇ CS enabled a comprehensive resistance and virulence analysis, providing a reliable method for bacterial strain typing and characterization of the virulome and resistome.

Carbapenemase-Encoding Genes and Colistin Resistance in Gram-Negative Bacteria During the COVID-19 Pandemic in Mexico: Results from the Invifar Network.

In this study, we report the carbapenemase-encoding genes and colistin resistance in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa in the second year of the COVID-19 pandemic. Clinical isolates included carbapenem-resistant K. pneumoniae, carbapenem-resistant E. coli, carbapenem-resistant A. baumannii, and carbapenem-resistant P. aeruginosa. Carbapenemase-encoding genes were detected by PCR. Carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates were analyzed using the Rapid Polymyxin NP assay. mcr genes were screened by PCR. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on representative isolates. A total of 80 carbapenem-resistant E. coli, 103 carbapenem-resistant K. pneumoniae, 284 carbapenem-resistant A. baumannii, and 129 carbapenem-resistant P. aeruginosa isolates were recovered. All carbapenem-resistant E. coli and carbapenem-resistant K. pneumoniae isolates were included for further analysis. A selection of carbapenem-resistant A. baumannii and carbapenem-resistant P. aeruginosa strains was further analyzed (86 carbapenem-resistant A. baumannii and 82 carbapenem-resistant P. aeruginosa). Among carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates, the most frequent gene was blaNDM (86/103 [83.5%] and 72/80 [90%], respectively). For carbapenem-resistant A. baumannii, the most frequently detected gene was blaOXA-40 (52/86, 60.5%), and for carbapenem-resistant P. aeruginosa, was blaVIM (19/82, 23.2%). For carbapenem-resistant A. baumannii, five indistinguishable pulsotypes were detected. Circulation of K. pneumoniae New Delhi metallo-ß-lactamase (NDM) and E. coli NDM was detected in Mexico. High virulence sequence types (STs), such as K. pneumoniae ST307, E. coli ST167, P. aeruginosa ST111, and A. baumannii ST2, were detected. Among K. pneumoniae isolates, 18/101 (17.8%) were positive for the Polymyxin NP test (two, 11.0% positive for the mcr-1 gene, and one, 5.6% with disruption of the mgrB gene). All E. coli isolates were negative for the Polymyxin NP test. In conclusion, K. pneumoniae NDM and E. coli NDM were detected in Mexico, with the circulation of highly virulent STs. These results are relevant in clinical practice to guide antibiotic therapies considering the molecular mechanisms of resistance to carbapenems.

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.

Increment Antimicrobial Resistance During the COVID-19 Pandemic: Results from the Invifar Network.

Aim: This study aims to assess the changes in antimicrobial resistance among some critical and high-priority microorganisms collected previously and during the coronavirus disease 2019 (COVID-19) pandemic in Mexico. Methods: We collected antimicrobial susceptibility data for critical and high-priority microorganisms from blood, urine, respiratory samples, and from all specimens, in which the pathogen may be considered a causative agent. Data were stratified and compared for two periods: 2019 versus 2020 and second semester 2019 (prepandemic) versus the second semester 2020 (pandemic). Results: In the analysis of second semester 2019 versus the second semester 2020, in blood samples, increased resistance to oxacillin (15.2% vs. 36.9%), erythromycin (25.7% vs. 42.8%), and clindamycin (24.8% vs. 43.3%) (p ≤ 0.01) was detected for Staphylococcus aureus, to imipenem (13% vs. 23.4%) and meropenem (11.2% vs. 21.4) (p ≤ 0.01), for Klebsiella pneumoniae. In all specimens, increased ampicillin and tetracycline resistance was detected for Enterococcus faecium (p ≤ 0.01). In cefepime, meropenem, levofloxacin, and gentamicin (p ≤ 0.01), resistance was detected for Escherichia coli; and in piperacillin-tazobactam, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, and gentamicin (p ≤ 0.01), resistance was detected for Pseudomonas aeruginosa. Conclusion: Antimicrobial resistance increased in Mexico during the COVID-19 pandemic. The increase in oxacillin resistance for S. aureus and carbapenem resistance for K. pneumoniae recovered from blood specimens deserves special attention. In addition, an increase in erythromycin resistance in S. aureus was detected, which may be associated with high azithromycin use. In general, for Acinetobacter baumannii and P. aeruginosa, increasing resistance rates were detected.

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.

Klebsiella variicola Reference Strain F2R9 (ATCC BAA-830) Genome Sequence.

Klebsiella variicola F2R9 was isolated from banana root, and its sequence has been deposited as ATCC BAA-830. It corresponds to sequence type 11 (ST11) and KL16 and contains no identifiable plasmids. The genome showed few antimicrobial resistance and virulence genes and several plant association genes. The strain showed susceptibility to most antimicrobials and avirulent behavior.

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 investigation of an outbreak associated with total parenteral nutrition contaminated with NDM-producing Leclercia adecarboxylata.

BACKGROUND: This study aimed to determine the epidemiological, microbiological, and molecular characteristics of an outbreak of carbapenem-resistant Leclercia adecarboxylata in three hospitals associated with the unintended use of contaminated total parental nutrition (TPN). METHODS: For 10 days, 25 patients who received intravenous TPN from the same batch of a formula developed sepsis and had blood cultures positive for L. adecarboxylata. Antimicrobial susceptibility and carbapenemase production were performed in 31 isolates, including one from an unopened bottle of TPN. Carbapenemase-encoding genes, extended-spectrum ß-lactamase-encoding genes were screened by PCR, and plasmid profiles were determined. Horizontal transfer of carbapenem resistance was performed by solid mating. Clonal diversity was performed by pulsed-field gel electrophoresis. The resistome was explored by whole-genome sequencing on two selected strains, and comparative genomics was performed using Roary. RESULTS: All 31 isolates were resistant to aztreonam, cephalosporins, carbapenems, trimethoprim/sulfamethoxazole, and susceptible to gentamicin, tetracycline, and colistin. Lower susceptibility to levofloxacin (51.6%) and ciprofloxacin (22.6%) was observed. All the isolates were carbapenemase producers and positive for blaNDM-1, blaTEM-1B, and blaSHV-12 genes. One main lineage was detected (clone A, 83.9%; A1, 12.9%; A2, 3.2%). The blaNDM-1 gene is embedded in a Tn125-like element. Genome analysis showed genes encoding resistance for aminoglycosides, quinolones, trimethoprim, colistin, phenicols, and sulphonamides and the presence of IncFII (Yp), IncHI2, and IncHI2A incompatibility groups. Comparative genomics showed a major phylogenetic relationship among L. adecarboxylata I1 and USDA-ARS-USMARC-60222 genomes, followed by our two selected strains. CONCLUSION: We present epidemiological, microbiological, and molecular evidence of an outbreak of carbapenem-resistant L. adecarboxylata in three hospitals in western Mexico associated with the use of contaminated TPN.

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.