Acinetobacter baumannii IC2 and IC5 Isolates with Co-Existing blaOXA-143-like and blaOXA-72 and Exhibiting Strong Biofilm Formation in a Mexican Hospital.

Acinetobacter baumannii is an opportunistic pathogen responsible for healthcare-associated infections (HAIs) and outbreaks. Antimicrobial resistance mechanisms and virulence factors allow it to survive and spread in the hospital environment. However, the molecular mechanisms of these traits and their association with international clones are frequently unknown in low- and middle-income countries. Here, we analyze the phenotype and genotype of seventy-six HAIs and outbreak-causing A. baumannii isolates from a Mexican hospital over ten years, with special attention to the carbapenem resistome and biofilm formation. The isolates belonged to the global international clone (IC) 2 and the Latin America endemic IC5 and were predominantly extensively drug-resistant (XDR). Oxacillinases were identified as a common source of carbapenem resistance. We noted the presence of the blaOXA-143-like family (not previously described in Mexico), the blaOXA-72 and the blaOXA-398 found in both ICs. A low prevalence of efflux pump overexpression activity associated with carbapenem resistance was observed. Finally, strong biofilm formation was found, and significant biofilm-related genes were identified, including bfmRS, csuA/BABCDE, pgaABCD and ompA. This study provides a comprehensive profile of the carbapenem resistome of A. baumannii isolates belonging to the same pulse type, along with their significant biofilm formation capacity. Furthermore, it contributes to a better understanding of their role in the recurrence of infection and the endemicity of these isolates in a Mexican hospital.

Gram-negative ESKAPE bacteria bloodstream infections in patients during the COVID-19 pandemic.

Bloodstream infections due to bacteria are a highly consequential nosocomial occurrences and the organisms responsible for them are usually multidrug-resistant. The aims of this study were to describe the incidence of bacteremia caused by Gram-negative ESKAPE bacilli during the COVID-19 pandemic and characterize the clinical and microbiological findings including antimicrobial resistance. A total of 115 Gram-negative ESKAPE isolates were collected from patients with nosocomial bacteremia (18% of the total bacteremias) in a tertiary care center in Mexico City from February 2020 to January 2021. These isolates were more frequently derived from the Respiratory Diseases Ward (27), followed by the Neurosurgery (12), Intensive Care Unit (11), Internal Medicine (11), and Infectious Diseases Unit (7). The most frequently isolated bacteria were Acinetobacter baumannii (34%), followed by Klebsiella pneumoniae (28%), Pseudomonas aeruginosa (23%) and Enterobacter spp (16%). A. baumannii showed the highest levels of multidrug-resistance (100%), followed by K. pneumoniae (87%), Enterobacter spp (34%) and P. aeruginosa (20%). The bla CTX-M-15 and bla TEM-1 genes were identified in all beta-lactam-resistant K. pneumoniae (27), while bla TEM-1 was found in 84.6% (33/39) of A. baumannii isolates. The carbapenemase gene bla OXA-398 was predominant among carbapenem-resistant A. baumannii (74%, 29/39) and bla OXA-24was detected in four isolates. One P. aeruginosa isolate was bla VIM-2 gene carrier, while two K. pneumoniae and one Enterobacter spp were bla NDM gene carriers. Among colistin-resistant isolates mcr-1 gene was not detected. Clonal diversity was observed in K. pneumoniae, P. aeruginosa and Enterobacter spp. Two outbreaks caused by A. baumannii ST208 and ST369 were detected, both belonging to the clonal complex CC92 and IC2. A. baumannii was associated with a death rate of 72% (28/32), most of them (86%, 24/28) extensively drug-resistant or pandrug-resistant isolates, mainly in patients with COVID-19 (86%, 24/28) in the Respiratory Diseases Ward. A. baumannii isolates had a higher mortality rate (72%), which was higher in patients with COVID-19. There was no statistically significant association between the multidrug-resistant profile in Gram-negative ESKAPE bacilli and COVID-19 disease. The results point to the important role of multidrug-resistant Gram-negative ESKAPE bacteria causing bacteremia in nosocomial settings before and during the COVID-19 epidemic. Additionally, we were unable to identify a local impact of the COVID-19 pandemic on antimicrobial resistance rates, at least in the short term.

Burkholderia vietnamiensis causing infections in noncystic fibrosis patients in a tertiary care hospital in Mexico.

Burkholderia cepacia complex (Bcc) species are opportunistic pathogens widely distributed in the environment and often infect people with cystic fibrosis (CF). This study aims to determine which genomovars of the Bcc can cause infections in non-CF patients from a tertiary care hospital in Mexico and if they carry virulence factors that could increase their pathogenicity. We identified 23 clinical isolates that carry the recA gene. Twenty-two of them belongs to the genomovar V (B. vietnamiensis) and one to the genomovar II (B. multivorans). Thirteen pulsotypes were identified among 22 B. vietnamiensis isolates. All clinical isolates produced biofilm were motile and cytotoxic on murine macrophage-like RAW264.7 and in A549 human lung epithelial cells. In conclusion, B. vietnamiensis causes infections in non-CF patients in a tertiary care hospital in Mexico, rapid identification of this pathogen can help physicians to establish a better antimicrobial treatment.

Pandrug-resistant Acinetobacter baumannii from different clones and regions in Mexico have a similar plasmid carrying the blaOXA-72 gene.

Background: Multidrug-resistant Acinetobacter baumannii is a common hospital-acquired pathogen. The increase in antibiotic resistance is commonly due to the acquisition of mobile genetic elements carrying antibiotic resistance genes. To comprehend this, we analyzed the resistome and virulome of Mexican A. baumannii multidrug-resistant isolates. Methods: Six clinical strains of A. baumannii from three Mexican hospitals were sequenced using the Illumina platform, the genomes were assembled with SPAdes and annotated with Prokka. Plasmid SPAdes and MobRecon were used to identify the potential plasmid sequences. Sequence Type (ST) assignation under the MLST Oxford scheme was performed using the PubMLST database. Homologous gene search for known virulent factors was performed using the virulence factor database VFDB and an in silico prediction of the resistome was conducted via the ResFinder databases. Results: The six strains studied belong to different STs and clonal complexes (CC): two strains were ST208 and one was ST369; these two STs belong to the same lineage CC92, which is part of the international clone (IC) 2. Another two strains were ST758 and one was ST1054, both STs belonging to the same lineage CC636, which is within IC5. The resistome analysis of the six strains identified between 7 to 14 antibiotic resistance genes to different families of drugs, including beta-lactams, aminoglycosides, fluoroquinolones and carbapenems. We detected between 1 to 4 plasmids per strain with sizes from 1,800 bp to 111,044 bp. Two strains from hospitals in Mexico City and Guadalajara had a plasmid each of 10,012 bp pAba78r and pAba79f, respectively, which contained the bla OXA-72 gene. The structure of this plasmid showed the same 13 genes in both strains, but 4 of them were inverted in one of the strains. Finally, the six strains contain 49 identical virulence genes related to immune response evasion, quorum-sensing, and secretion systems, among others. Conclusion: Resistance to carbapenems due to pAba78r and pAba79f plasmids in Aba pandrug-resistant strains from different geographic areas of Mexico and different clones was detected. Our results provide further evidence that plasmids are highly relevant for the horizontal transfer of antibiotic resistance genes between different clones of A. baumannii.

Contributions and difficulties of soil metagenomics and its impact on agricultura / Aportes y dificultades de la metagenómica de suelos y su impacto en la agricultura

RESUMEN Los microorganismos son de gran interés porque colonizan todo tipo de ambiente, sin embargo, uno de los problemas al que nos enfrentamos para conocer su diversidad biológica es que no todos los microorganismos son cultivables. El desarrollo de nuevas tecnologías como la generación de vectores de clonación aunado al desarrollo de técnicas de secuenciación de alto rendimiento ha favorecido el surgimiento de una nueva herramienta llamada metagenómica, la cual nos permite estudiar genomas de comunidades enteras de microorganismos. Debido a que ningún ambiente es idéntico a otro, es importante mencionar que dependiendo del tipo de muestra a analizar será el tipo de reto al cual nos enfrentaremos al trabajar con metagenómica, en el caso específico del suelo existen diversas variantes como la contaminación del suelo con metales pesados o diversos compuestos químicos que podrían limitar los estudios. Sin embargo, pese a las limitaciones que el mismo ambiente presenta, la metagenómica ha permitido tanto el descubrimiento de nuevos genes como la caracterización de las comunidades microbianas que influyen positivamente en el desarrollo de plantas, lo cual en un futuro podría generar un gran impacto en la agricultura. En este artículo se realizó una revisión de diversas investigaciones que han empleado metagenómica, reportadas en las bases de datos de PudMed y Google Schoolar, con el objetivo de examinar los beneficios y limitaciones de las diversas metodologías empleadas en el tratamiento del ADN metagenómico de suelo y el impacto de la metagenómica en la agricultura.

ABSTRACT Microorganisms are of great interest because they colonize all types of environment, however, one of the problems we face in knowing biological diversity is that not all microorganisms are cultivable. The development of new technologies such as the generation of cloning vectors coupled with the development of high performance sequencing techniques, have favored the emergence of a new tool in science called metagenomics, which allows us to study genomes of entire communities. Since all environments are different, the type of challenge that we will face when working with metagenomics is going to change depending of the type of sample, in the specific case of soils, there are several variables, such as soil contamination with heavy metals or chemical compounds that could limit metagenomic studies. However, despite the limitations that the environment presents, with the help of metagenomics, both gene discovery and the characterization of microbial communities that positively influence plant development have been achieved, which could generate a greater impact on agriculture in the future. In this article a review of several investigations that have used metagenomics, reported in the PudMed and Google Schoolar databases was carried out, with the aim of examining the benefits and limitations of the various methodologies used in the treatment of metagenomic DNA from soil and the impact of metagenomics in agriculture.

Accessory Genomic Epidemiology of Cocirculating Acinetobacter baumannii Clones.

Acinetobacter baumannii has become one of the most important multidrug-resistant nosocomial pathogens all over the world. Nonetheless, very little is known about the diversity of A. baumannii lineages coexisting in hospital settings. Here, using whole-genome sequencing, epidemiological data, and antimicrobial susceptibility tests, we uncover the transmission dynamics of extensive and multidrug-resistant A. baumannii in a tertiary hospital over a decade. Our core genome phylogeny of almost 300 genomes suggests that there were several introductions of lineages from international clone 2 into the hospital. The molecular dating analysis shows that these introductions happened in 2006, 2007, and 2013. Furthermore, using the accessory genome, we show that these lineages were extensively disseminated across many wards in the hospital. Our results demonstrate that accessory genome variation can be a very powerful tool for conducting genomic epidemiology. We anticipate future studies employing the accessory genome along with the core genome as a powerful phylogenomic strategy to track bacterial transmissions over very short microevolutionary scales. IMPORTANCE Whole-genome sequencing for epidemiological investigations (genomic epidemiology) has been of paramount importance to understand the transmission dynamics of many bacterial (and nonbacterial) pathogens. Commonly, variation in the core genome, single nucleotide polymorphisms (SNPs), is employed to carry out genomic epidemiology. However, at very short periods of time, the core genome might not have accumulated enough variation (sufficient SNPs) to tell apart isolates. In this scenario, gene content variation in the accessory genome can be an option to conduct genomic epidemiology. Here, we used the accessory genome, as well as the core genome, to uncover the transmission dynamics of extensive and multidrug-resistant A. baumannii in a tertiary hospital for a decade. Our study shows that accessory genome variation can be a very powerful tool for conducting genomic epidemiology.

Dissemination of bla NDM- 1 Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.

Nosocomial infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are a major health problem worldwide. The aim of this study was to describe NDM-1-producing K. pneumoniae strains causing bacteremia in a tertiary referral hospital in Mexico. MDR K. pneumoniae isolates were screened by polymerase chain reaction for the presence of resistance genes. In resistant isolates, plasmids were identified and conjugation assays were performed. Clonal diversity and the sequence types were determined by pulsed-field gel electrophoresis and multilocus sequence typing. A total of 80 K. pneumoniae isolates were collected from patients with bacteremia over a 1-year period. These isolates showed a level of resistance of 59% (47/80) to aztreonam, 56-60% (45-48/80) to cephalosporins, 54% (43/80) to colistin and 12.5% (10/80) to carbapenems. The carbapenem resistant isolates were bla NDM- 1 carriers and negative for bla KPC, bla NDM, bla IMP, bla VIM and bla OXA- 48 -like carbapenemases genes. Conjugative plasmids IncFIIA and IncF group with sizes of 82-195 kbp were carriers of bla NDM- 1, bla CTX-M- 15, bla TEM- 1, aac(6')-Ib and/or aac(3')-IIa. Clonal variability and nine different multilocus sequence types were detected (ST661, ST683, ST1395, ST2706, ST252, ST1198, ST690, ST1535, and ST3368) for the first time in the isolates carrying bla NDM- 1 in Mexico. This study demonstrates that bla NDM- 1 has remained within this hospital in recent years and suggests that it is currently the most prevalent carbapenemase among K. pneumoniae MDR strains causing bacteremia in Mexico. The horizontal transfer of bla NDM- 1 gene through IncF-like plasmids among different clones demonstrates the dissemination pathway of antimicrobial resistance and underscore the need for strong and urgent joint measures to control the spread of NDM-1 carbapenemase in the hospital.

Carbapenem-Resistant Acinetobacter baumannii in Three Tertiary Care Hospitals in Mexico: Virulence Profiles, Innate Immune Response and Clonal Dissemination.

Acinetobacter baumannii is one of the most important nosocomial pathogens distributed worldwide. Due to its multidrug-resistance and the propensity for the epidemic spread, the World Health Organization includes this bacterium as a priority health issue for development of new antibiotics. The aims of this study were to investigate the antimicrobial resistance profile, the clonal relatedness, the virulence profiles, the innate host immune response and the clonal dissemination of A. baumannii in Hospital Civil de Guadalajara (HCG), Hospital Regional General Ignacio Zaragoza (HRGIZ) and Pediatric ward of the Hospital General de México Eduardo Liceaga (HGM-P). A total of 252 A. baumannii clinical isolates were collected from patients with nosocomial infections in these hospitals between 2015 and 2016. These isolates showed a multidrug-resistant profile and most of them only susceptible to colistin. Furthermore, 83.3 and 36.9% of the isolates carried the bla OXA- 24 and bla TEM- 1 genes for resistance to carbapenems and ß-lactam antibiotics, respectively. The clonal relatedness assessed by pulsed-field gel electrophoresis (PFGE) and by multi-locus sequence typing (MLST) demonstrated a genetic diversity. Remarkably, the ST136, ST208 and ST369 that belonged to the clonal complex CC92 and ST758 and ST1054 to the CC636 clonal complex were identified. The ST136 was a high-risk persistent clone involved in an outbreak at HCG and ST369 were related to the first carbapenem-resistant A. baumannii outbreak in HRGIZ. Up to 58% isolates were able to attach to A549 epithelial cells and 14.5% of them induced >50% of cytotoxicity. A549 cells infected with A. baumannii produced TNFα, IL-6 and IL-1ß and the oxygen and nitrogen reactive species that contributes to the development of an inflammatory immune response. Up to 91.3% of clinical isolates were resistant to normal human serum activity. Finally, 98.5% of the clinical isolates were able to form biofilm over polystyrene tubes. In summary, these results demonstrate the increasingly dissemination of multidrug-resistant A. baumannii clones in three hospitals in Mexico carrying diverse bacterial virulence factors that could contribute to establishment of the innate immune response associated to the fatality risks in seriously ill patients.

Emergence of IncFIA Plasmid-Carrying blaNDM-1 Among Klebsiella pneumoniae and Enterobacter cloacae Isolates in a Tertiary Referral Hospital in Mexico.

The emergence of New Delhi metallo-ß-lactamase 1 on carbapenemase-producing bacteria has raised a major worldwide public health concern. This study reports the dissemination of blaNDM-1 in carbapenem-resistant isolates that caused nosocomial infections in a tertiary hospital in Mexico City. Seven Enterobacter cloacae and three Klebsiella pneumoniae nosocomial isolates from the same time period harbored the blaNDM-1 gene. The resistance phenotype and the blaNDM-1 gene were transferred through conjugative plasmids belonging to the incompatibility group IncFIA of 85, 101, and 195 kb in E. cloacae and 95 and 101 kb in K. pneumoniae isolates. Restriction fragment length polymorphism analysis showed that blaNDM-1 was carried in similar plasmids with molecular sizes of 101 and 85 kb, each one in three isolates of E. cloacae and one of 101 kb on two isolates of K. pneumoniae. During a 9-month period, six of the seven isolates of E. cloacae analyzed harbored blaNDM-1 and belonged to clone E1. Similarly, over a 5-month period, two of the three K. pneumoniae isolates that harbored blaNDM-1 belonged to clone K1. These results demonstrate the horizontal transfer of blaNDM-1 between different bacterial species, dissemination of clones with high levels of resistance to carbapenems, and underscore the need for heightened measures to control their further spread.

Virulence profiles and innate immune responses against highly lethal, multidrug-resistant nosocomial isolates of Acinetobacter baumannii from a tertiary care hospital in Mexico.

Virulence profiles and innate immune responses were studied in Acinetobacter baumannii from nosocomial infections collected over one year in a tertiary care hospital in Mexico. A. baumannii were identified by VITEK 2 System followed by susceptibility tests. Carbapenemase genes, active efflux mechanism to imipenem and meropenem and outer membrane proteins profile were analyzed to evaluate their role on the activity of carbapenem resistance. All isolates were genotyped by pulsed field gel electrophoresis. The ability to form biofilm was determined on a polystyrene surface. The resistance to complement was determined with a pooled human normal serum and TNFα release by infected macrophages was determined by ELISA. The 112 isolates from this study were associated with a 52% of mortality. All were resistance to ß-lactams, fluoroquinolones, and trimethroprim-sulfamethoxal, 96 and 90% were resistant to meropenem and imipenem, respectively, but with high susceptibility to polymyxin B, colistin and tigecyclin. Isolates were classified in 11 different clones. Most isolates, 88% (99/112), were metallo-ß-lactamases and carbapenemases producers, associated in 95% with the presence of blaOXA-72 gene. Only 4/99 and 1/99 of the carbapenem-resistant isolates were related to efflux mechanism to meropenem or imipenem resistance, respectively. The loss of expression of 22, 29, and/or 33-36-kDa proteins was detected in 8/11 of the clinical isolates with resistance to carbapenem. More than 96% (108/112) of the isolates were high producers of biofilms on biotic surfaces. Finally, all isolates showed variable resistance to normal human serum activity and were high inductors of TNFα release by macrophages. In summary, these results suggest that multidrug-resistant A. baumannii can persist in the hospital environment through its ability to form biofilms. The high mortality observed was due to their ability to survive normal human serum activity and capability to induce potent inflammatory immune response making this nosocomial pathogen a serious threat to hospitalized patients.

Molecular mechanisms associated with nosocomial carbapenem-resistant Acinetobacter baumannii in Mexico.

BACKGROUND AND AIMS: Acinetobacter baumannii is an emerging pathogen worldwide that is most commonly associated with nosocomial infections and multi-drug resistance. In the present study we determined the mechanisms of carbapenem resistance and clonal diversity of A. baumannii nosocomial isolates in Hospital Civil de Guadalajara, Mexico. METHODS: A total of 303 clinical isolates of A. baumannii identified during a period expanding from 2004-2011 were analyzed for carbapenem resistance using several microbiological and molecular methods. Clonal relatedness of these isolates was determined using pulsed-field gel electrophoresis. RESULTS: Of the 303 isolates, 84% were resistant to meropenem, 71.3% to imipenem and 78.3% the resistant isolates were positive for metallo-ß-lactamases as determined by the phenotypic assay. In addition, 49.6% of carbapenem-intermediate or -resistant isolates carried the blaOXA-72 gene and 1.2% carried the blaVIM-1 gene. Efflux pump phenotype was responsible for reduced susceptibility to meropenem in 14.5% and to imipenem in 31.6% of the resistant isolates, respectively in the presence of the efflux pump inhibitor, carbonyl cyanide 3-chlorophenylhydrazone. Strains representing different carbapenem-resistant patterns exhibited reduced expression of 22, 29, 33, and 43 kDa OMPs. Among the bacterial collection studied, 48 different clones were identified, two of which were predominant and persistently transmitted. CONCLUSIONS: Carbapenemase production in combination with efflux pump expression, reduction in OMPs expression and the cross-transmission of clones appear to be major contributors to the high frequency of carbapenem-resistance observed in A. baumannii. To our knowledge, this is the first study to define the molecular mechanisms associated with carbapenem-resistance in A. baumannii in Mexico.

Role of the C-terminal region of dextransucrase from Leuconostoc mesenteroides IBT-PQ in cell anchoring.

dsrP, a gene that encodes a cell-associated dextransucrase produced by Leuconostoc mesenteroides IBT-PQ, was isolated, sequenced and expressed in Escherichia coli. From sequence analysis, seven repeat units in the N-terminal region were found, as well as five cell wall binding repeats in the C-terminal region. A model of the C-terminal domain of dextransucrase was built based on the solenoid structure of the cell wall binding domain already described in LytA. By experiments involving direct interactions of the enzyme with L. mesenteroides cells, as well as among the cells and the single C-terminal domain expressed in E. coli, evidence was obtained concerning the anchoring function of this region in cell-associated dextransucrase, a function which may be independent of its capacity to bind dextran.

Rapid evolutionary change of common bean (Phaseolus vulgaris L) plastome, and the genomic diversification of legume chloroplasts.

BACKGROUND: Fabaceae (legumes) is one of the largest families of flowering plants, and some members are important crops. In contrast to what we know about their great diversity or economic importance, our knowledge at the genomic level of chloroplast genomes (cpDNAs or plastomes) for these crops is limited. RESULTS: We sequenced the complete genome of the common bean (Phaseolus vulgaris cv. Negro Jamapa) chloroplast. The plastome of P. vulgaris is a 150,285 bp circular molecule. It has gene content similar to that of other legume plastomes, but contains two pseudogenes, rpl33 and rps16. A distinct inversion occurred at the junction points of trnH-GUG/rpl14 and rps19/rps8, as in adzuki bean 1. These two pseudogenes and the inversion were confirmed in 10 varieties representing the two domestication centers of the bean. Genomic comparative analysis indicated that inversions generally occur in legume plastomes and the magnitude and localization of insertions/deletions (indels) also vary. The analysis of repeat sequences demonstrated that patterns and sequences of tandem repeats had an important impact on sequence diversification between legume plastomes and tandem repeats did not belong to dispersed repeats. Interestingly, P. vulgaris plastome had higher evolutionary rates of change on both genomic and gene levels than G. max, which could be the consequence of pressure from both mutation and natural selection. CONCLUSION: Legume chloroplast genomes are widely diversified in gene content, gene order, indel structure, abundance and localization of repetitive sequences, intracellular sequence exchange and evolutionary rates. The P. vulgaris plastome is a rapidly evolving genome.