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.

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.

Interferon-tau (IFN-τ) Has Antiproliferative Effects, Induces Apoptosis, and Inhibits Tumor Growth in a Triple-negative Breast Cancer Murine Tumor Model.

BACKGROUND/AIM: Resistant triple-negative breast cancer (TNBC) is a subtype of this disease that is resistant to conventional chemotherapy agents. IFN-τ is a cytokine that has recently been shown to have immunoregulatory and antitumor effects. The present study aimed to examine the antiproliferative and apoptosis effects of IFN-τ in breast cancer cells and the antitumor effect in a murine tumor model of TNBC. MATERIALS AND METHODS: Murine breast cancer 4T1 cells were cultured and treated with ovine IFN-τ and through MTT and Caspase-Glo 3/7 assays, viability and cell death were determined. In addition, the antitumor effect of IFN-τ was determined in a murine tumor model of TNBC. RESULTS: Ovine IFN-τ showed a concentration-dependent antiproliferative effect on 4T1 murine breast cancer cells. Also, treatment of 4T1 cells with IFN-τ induced the activation of caspase 3 and 7, which is indicative of apoptotic cell death. Moreover, we detected an increase in the expression of type I interferon receptor (IFNAR1/2) in cells treated with IFN-. The intratumoral application of IFN-τ in mice inhibited tumor growth compared to the control non-treated group, and the effect was associated with the increased expression of GM-CSF. CONCLUSION: Ovine IFN-τ may be an effective immunotherapeutic cytokine for the treatment of TNBC.

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.

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.

Draft Genome Sequences of Endophytic Isolates of Klebsiella variicola and Klebsiella pneumoniae Obtained from the Same Sugarcane Plant.

Endophytic Klebsiella variicola KvMx2 and Klebsiella pneumoniae KpMx1 isolates obtained from the same sugarcane stem were used for whole-genome sequencing. The genomes revealed clear differences in essential genes for plant growth, development, and detoxification, as well as nitrogen fixation, catalases, cellulases, and shared virulence factors described in the K. pneumoniae pathogen.

ESBL-producing Escherichia coli and Klebsiella pneumoniae: The most prevalent clinical isolates obtained between 2005 and 2012 in Mexico.

OBJECTIVES: To identify the prevalence of ESBL genes in the principal group of Enterobacteriaceae causing nosocomial infections and to identify the phylogenetic group in Escherichia coli isolates. METHODS: There were collected 1084 ESBL-producing Enterobacteriaceae isolates during 2005-2012 from adult patients from 14 hospitals and corresponding to eight states and five regions (SE, S, N, W and NW) in Mexico. The CTX-M-(CTX-M-1 group), SHV-, TLA- and GES-type ESBLs genes were screened. The respective alleles were determined in the most of ESBLs genes. In E. coli isolates selected were used to identify the phylogenetic group. RESULTS: The ESBL-producing Escherichia coli and Klebsiella pneumoniae corresponded the most prevalent clinical isolates. CTX-M-type ESBLs genes were the most common, followed by SHV-type, GES-type and the ESBLs TLA-1 gene. The allelic frequency showed to CTX-M-15 ESBL the most prevalent, followed by the SHV-12, SHV-5 and GES-1, GES-19 in the GES family. Among ESBL-producing E. coli isolates the phylogenetic groups A and D were the most common ones. CONCLUSIONS: The present study showed an epidemiological change in terms of bacterial species, placing E. coli as the most frequently isolated bacteria among ESBL-producing Enterobacteriaceae in Mexico, followed by K. pneumoniae. This frequency is accompanied by a high frequency of ESBL CTX-M-15.

Molecular characterization of ESBL-producing Escherichia coli isolates from hospital- and community-acquired infections in NW Mexico.

We investigated the molecular characteristics of ESBL-producing E. coli (ESBL-PEc) isolates from two hospitals and community settings in Ciudad Obregon, Sonora, Mexico. Between 2011 and 2014, thirty-seven ESBL-PEc isolates were collected. The major encoded ESBL was the blaCTX-M-15 gene (97%); followed by 13.5% of the blaSHV-12 gene, and 5.5% encoded the blaTLA-1 gene. The PMQR gene aac(6´)-Ib-cr was detected in 97% of the isolates and the qnrB gene, in one isolate. The ESBL-PEc isolates corresponded to phylogenetic group B2, ST131. Our results highlight the dissemination of ESBL-PEc isolates in northwest Mexico (Ciudad Obregon, Sonora).

Outbreak Caused by blaOXA-72-Producing Acinetobacter baumannii ST417 Detected in Clinical and Environmental Isolates.

We characterized an outbreak of imipenem-resistant Acinetobacter baumannii with clinical and environmental isolates from a tertiary care hospital in San Luis Potosi, Mexico. During a 4-month period, a total of 32 nonrepetitive imipenem-resistant clinical isolates of A. baumannii were collected. All isolates were susceptible to colistin and tigecycline and resistant to cefepime, ceftazidime, ceftriaxone, imipenem, and meropenem. Genotyping by pulsed-field gel electrophoresis showed a major clone (A). Multilocus sequence type (MLST) analysis was performed, revealing sequence type (ST) 417 (ST417) and 208 (ST208). The blaIMP-, blaVIM-, blaGIM-, blaSIM-, blaNDM-type, and blaOXA-type (blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like) genes were screened and showed that the blaOXA-51-like and blaOXA-24-like genes were present in all isolates. Sequencing and southern hybridization were performed, confirming the presence of the blaOXA-72 gene and its plasmid-borne nature. In addition, the blaOXA-72-XerC/XerD-like association was identified. These findings indicate that a clonal spread of blaOXA-72-producing A. baumannii ST417 had occurred throughout the hospital. The ST417 corresponded with a previous ST described in the United States.

Identification and Characterization of Imipenem-Resistant Klebsiella pneumoniae and Susceptible Klebsiella variicola Isolates Obtained from the Same Patient.

Klebsiella variicola, a bacterium closely genetically related to Klebsiella pneumoniae, is commonly misidentified as K. pneumoniae by biochemical tests. To distinguish between the two bacteria, phylogenetic analysis of the rpoB gene and the identification of unique genes in both bacterial species by multiplex-polymerase chain reaction (PCR) provide the means to reliably identify and genotype K. variicola. In recent years, K. variicola has been described both as the cause of an intrahospital outbreak in a pediatric hospital, which resulted in sepsis in inpatients, and as a frequent cause of bloodstream infections. In the present study, K. pneumoniae and K. variicola were isolated from a unique patient displaying different antimicrobial susceptibility phenotypes and different genotypes of virulence determinants. Eight clinical isolates were obtained at different time intervals; all during a 5-month period. The isolates were identified as K. pneumoniae by an automated identification system. The clinical (biochemical test) and molecular (multiplex-PCR and rpoB gene) characterization identified imipenem resistance in the first six K. pneumoniae ST258 isolates, which encode the SHV-12 cephalosporinase and KPC-3 carbapenemase genes. The two last remaining isolates corresponded to susceptible K. variicola. The bacterial species showed a specific profile of virulence-associated determinants, specifically the fimA, fimH, and ecpRAB fimbrial-encoding genes identified only in K. pneumoniae isolates. However, the entb (enterobactin), mrkD (fimbrial adhesin), uge (epimerase), ureA (urease), and wabG (transferase) genes were shared between both bacterial species. Recent studies attribute a higher mortality rate to K. variicola than to K. pneumonia. This work highlights the identification of K. pneumoniae and the closely related K. variicola isolated from the same patient. The value of distinguishing between these two bacterial species is in their clinical significance, their different phenotypes and genotypes, and the fact that they can be isolated from the same patient.

Widespread of ESBL- and carbapenemase GES-type genes on carbapenem-resistant Pseudomonas aeruginosa clinical isolates: a multicenter study in Mexican hospitals.

The present work describes a prevalence of 36.2% of carbapenemases IMP-, VIM-, and GES-type on 124 imipenem-resistant Pseudomonas aeruginosa clinical isolates. The ESBL GES-19 and carbapenemase GES-20 genes were the most prevalent (84.4%) ß-lactamases among imipenem-resistant P. aeruginosa clinical isolates in Mexico. These genes are chromosomal encoded on embedded class 1 integron arrays.

Characteristics of KPC-2-producing Klebsiella pneumoniae (ST258) clinical isolates from outbreaks in 2 Mexican medical centers.

The KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) is an important pathogen widely spread in nosocomial infections. In this study, we identified the KPC-2-producing K. pneumoniae clinical isolates of 2 unrelated outbreaks that corresponded to pandemic strain ST258. The isolates showed high resistance to cephalosporins, carbapenems, quinolones, and colistin. The KPC-2-producing K. pneumoniae isolates were compared to the previously studied KPC-3-producing K. pneumoniae isolates from an outbreak in Mexico; they showed an unrelated pulsed-field gel electrophoresis fingerprinting pattern and a different plasmid profile. The KPC-2 carbapenemase gene was identified in two 230- and 270-kb non-conjugative plasmids; however, 1 isolate transferred the KPC-2 gene onto an 80-kb plasmid. These findings endorse the need of carrying out a continuous molecular epidemiological surveillance of carbapenem-resistant isolates in hospitals in Mexico.

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion.

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Characterization of plasmid-mediated quinolone resistance (PMQR) genes in extended-spectrum ß-lactamase-producing Enterobacteriaceae pediatric clinical isolates in Mexico.

This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR) genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17) corresponded to E. cloacae, 13.7% (7/51) to K. pneumoniae, and 13.6% (6/44) to E. coli. In addition, the prevalence of aac(6')-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum ß-lactamase (ESBL) CTX-M-15 as the most prevalent one (70.5%), and to SHV-12 in the case of aac(6')-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of bla(CTX-M-15) with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6')-Ib-cr) presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6')-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.

Molecular epidemiology of Escherichia coli O25b-ST131 isolates causing community-acquired UTIs in Mexico.

Escherichia coli is a common uropathogen causing community-acquired urinary tract infections (UTIs). Out of 4735 E. coli community-acquired UTIs, 10.2% were extended spectrum ß-lactamases (ESBL)-producing. The identified ESBL types were CTX-M-15 (96.4%), SHV-2a (3%), and TLA-1 (1%). Of the isolates, 94.6% tested positive for plasmid-mediated quinolone resistance (PMQR) genes (aac(6')-lb-cr [92.1%] and qepA1 [7%] and for qnr-determinants [3.5%]). E. coli O25b-ST131 was identified in 25% of the isolates that harbor a non-conjugative 160-kb plasmid (IncFIA) containing the CTX-M-15, and all of these isolates were found to contain PMQR genes. This work can be useful in modeling the potential impact that may have on community-acquired UTIs in Mexico.

Characterization of Enterobacteriaceae isolates obtained from a tertiary care hospital in Mexico, which produces extended-spectrum ß-lactamase.

The prevalence and genetic characteristics of Escherichia coli and Klebsiella pneumoniae clinical isolates producing extended-spectrum ß-lactamase (ESBL) were examined. Between October 2010 and March 2011, E. coli (n=460) and K. pneumoniae (n=78) isolates were collected at a tertiary care hospital in Guadalajara, Mexico. The minimum inhibitory concentration (MIC) for each isolate was determined using a broth microdilution method, and ESBL production was assayed. The presence of ß-lactamase genes, blaSHV, blaCTX-M, and blaTLA-1, was detected by PCR and confirmed with sequencing. Only ESBL-producing isolates were further subjected to pulsed-field gel electrophoresis (PFGE) and plasmid profiling. All of the ESBL isolates were multidrug resistant and 75/460 (16.3%) E. coli isolates and 21/78 (26.9%) K. pneumoniae isolates were found to produce ESBL. For the E. coli isolates, >95% susceptibility to amikacin, meropenem, fosfomycin, imipenem, and nitrofurantoin was observed. For K. pneumoniae, similar results were obtained, with discrepancies observed for gentamicin and nitrofurantoin. PFGE further identified eleven pulsotypes for E. coli and three clusters of K. pneumoniae. CTX-M-15 was detected in 85% of ESBL-producing E. coli and in 76% of ESBL-producing K. pneumoniae. In contrast, SHV-5 ESBL was identified in 17% of E. coli isolates and in 86% of K. pneumoniae isolates. The bla-TLA-1 gene was not detected in any of the 96 isolates analyzed. Overall, CTX-M-15 and SHV-5 were found to have a high rate of spread throughout the hospital and were associated with strong multidrug resistance.

Transfer of quinolone resistance gene qnrA1 to Escherichia coli through a 50 kb conjugative plasmid resulting from the splitting of a 300 kb plasmid.

OBJECTIVES: To analyse the in vitro transfer of the qnrA1 gene by a 50 kb (pSZ50) self-transferable plasmid that derives from a 300 kb plasmid (pSZ300) and to determine the complete nucleotide sequence of plasmid pSZ50. METHODS: Extended-spectrum ß-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes of an Escherichia coli clinical isolate were analysed. Plasmid analysis included conjugation and selection on seven antibiotics examined by antimicrobial susceptibility testing, RFLP comparison, Southern hybridization, incompatibility group identification and shotgun sequencing. RESULTS: The E. coli 5509 isolate carries the genes encoding the ESBL CTX-M-15 and the quinolone resistance determinants qnrA1, qnrB2 and aac(6')-Ib-cr on a 300 kb plasmid. Seven transfer resistances were analysed by conjugation under two conditions (30 and 37°C), leading to two distinct transconjugant phenotypes with different resistances. Transconjugants of phenotype A harboured a 300 kb plasmid named pSZ300 that conferred resistance to eight antibiotics and harboured the qnrA1, aac(6')-Ib-cr and bla(CTX-M-15) genes. Transconjugants of phenotype B were resistant to three antibiotics and they harboured the qnrA1 gene on an ≈ 50 kb plasmid named pSZ50. Both plasmids were self-transferable at a frequency of 1 × 10(-3). Plasmid pSZ300 was typed to be both an IncF and IncN plasmid, whereas pSZ50 corresponded only to type IncN. Fingerprinting and Southern hybridization showed that plasmid pSZ50 derived from pSZ300. The complete nucleotide sequence of plasmid pSZ50 was determined (51556 bp) and 55 open reading frames were predicted. The qnrA1 gene was identified in a tandem duplicate inside a sul1-type integron structure. CONCLUSIONS: The plasmid pSZ300 represented a fusion of two replicons (IncF and IncN), and our observations suggest that the plasmid pSZ50 (IncN) may split and transfer antibiotic resistance determinants. This mechanism could be advantageous in the dissemination of antibiotic resistance genes.