Draft genome sequence of uropathogenic Escherichia coli U13824, a multidrug-resistant (MDR) and extended-spectrum-ß-lactamase (ESBL)-producing UPEC strain isolated from an adult woman with urinary tract infection.

Urinary tract infections (UTIs) caused by multidrug-resistant and extended-spectrum ß-lactamase-producing uropathogenic Escherichia coli are a worldwide concern. We report the draft genome of E. coli U13824 isolated from a female outpatient with UTI. This genome's availability strengthens the genomic surveillance of antimicrobial resistance and the spreading of these strains.

Complete mitochondrial genome of Gnathostoma binucleatum.

This report describes the mitochondrial genome of the parasite Gnathostoma binucleatum (G. binucleatum), which was obtained from naturally infected freshwater fish in Sinaloa, Mexico (22°46'00.1″N 105°40'21.8″W). G. binucleatum is responsible for human gnathostomiasis and is endemic to Mexico. It belongs to the Spirurida order of the Secernentea class of Nematoda.

Multidrug resistance and class 1 integron presence in Escherichia coli isolates from a polluted drainage ditch's water.

The impact of contamination of water drainage ditches in the development of antibiotic-resistant bacteria has been scarcely studied in Mexico. In this regard, 101 isolates of E. coli were obtained from water samples from a ditch in Sinaloa, during one year. The antimicrobial resistant profiles, the presence of the class 1 integron and evolutionary relationship of intI1 sequences were determined. The 47.5% of strains were resistant and 5.9% multidrug resistant (MDR) with an average multiple antibiotic resistance index value of 0.45. The highest resistance was registered with ß-lactam (39.6%) and quinolone (9.9%). The intI1 gene was detected in 11.9% of the isolates, and no association with MDR was found. Sequence were associated with human and animal host isolates. MDR E. coli isolates with intI1 gene highlight the potential risk of the ditch's water to human health. An attenuation effect of MDR E. coli isolates in the outlet water was observed.

mRNA levels of tricarboxylic acid cycle genes in Streptomyces coelicolor M145 cultured on glucose.

BACKGROUND: Streptomyces strains degrade many complex organic compounds and produce secondary metabolites. In aerobic organisms such as Streptomyces species, the tricarboxylic acid (TCA) cycle represents an indispensable central carbon metabolic pathway for energy generation and metabolic intermediary replenishment. Although various precursors for antibiotic biosynthesis are derived from this cycle, relatively few studies have focused on determining how a single carbon source can impact this metabolic pathway at different growth phases. In this study, we identified chromosomal genes involved in the TCA cycle in Streptomyces coelicolor and determined their mRNA levels. METHODS AND RESULTS: We searched the genes involved in the TCA cycle in S. coelicolor through bioinformatic analysis. Growth, glucose concentration quantification and RNA isolation were made from cultures of S. coelicolor grown on minimal medium with glucose along 72 h. mRNA levels of all identified genes were obtained by RT-qPCR. Five enzymes encoded by a single gene each were found, while for the rest at least two genes were found. The results showed that all the genes corresponding to the TCA enzymes were transcribed at very different levels and some of them displayed growth-phase dependent expression. CONCLUSION: All TCA cycle-associated genes, including paralog genes, were differentially transcribed in S. coelicolor grown in minimal medium with glucose as carbon source. Some of them, such as succinyl-CoA synthetase and succinate dehydrogenase, have low mRNA levels, which could limit the carbon flux through the TCA cycle. Our findings suggest that the genetic expansion of TCA cycle genes could confer to S. coelicolor the ability to adapt to diverse nutritional conditions and metabolic changes through different paralog genes expression.

Effect of 4-hydroxychalcone as preventive and curative treatment in Wistar rats with liver injury.

The increasing prevalence and complications related to liver diseases (caused by infection, toxic agents, or metabolic syndrome), together with insufficient existence of treatments, make evident the need for better therapeutic alternatives. Therefore, the aim of this study was to determine the effect of 4-hydroxychalcone (4-HC) as preventive and curative treatment in acute and chronic liver injury, respectively. Liver damage was induced with carbon tetrachloride (CCl4) in Wistar rats. Rats were divided into two groups: (1) acute liver injury and (2) chronic liver injury. In turn, each group was divided into four subgroups: (i) control (water); (ii) dimethyl sulfoxide 10%; (iii) CCl4; and (iv) 4-HC. The pre-treatment with 4-HC decreased transaminases, IL-6 serum levels, and hepatic malondialdehyde, increased IL-10 serum levels and hepatic glutathione, and decreased liver damage (necrosis, steatosis, and inflammatory infiltrate). In contrast, treatment with 4-HC after the induction of chronic liver injury decreased IL-6 serum levels and liver damage (steatosis, inflammatory infiltrate, ballooning cells, steatofibrosis, and fibrosis degree). Thus, the 4-HC treatment is proposed as a preventive treatment against acute liver injury; moreover, these results suggested the potential of 4-HC as a curative treatment against chronic liver injury, but other scheme treatments must be evaluated in future.

Genomic Profiling of Antibiotic-Resistant Escherichia coli Isolates from Surface Water of Agricultural Drainage in North-Western Mexico: Detection of the International High-Risk Lineages ST410 and ST617.

Aquatic environments are recognized as one of the main reservoirs for the emergence and dissemination of high-risk lineages of multidrug-resistant (MDR) bacteria of public health concern. However, the genomic characteristics of antibiotic-resistant Escherichia coli isolates from aquatic origins remain limited. Herein, we examined the antibiotic resistance and virulence genomic profiles of three E. coli recovered from surface water in northwest Mexico. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), and in-depth in silico analysis were performed. Two E. coli exhibited MDR phenotypes. WGS-based typing revealed genetic diversity, and phylogenetic analysis corroborated a notable divergent relationship among the studied E. coli. One E. coli strain, harboring enterotoxigenic and extraintestinal pathogenic-associated virulence genes, was assigned to the ST4 lineage. MDR E. coli, belonging to the international high-risk clones ST410 and ST617, carried genes and mutations conferring resistance to aminoglycosides, ß-lactams, quinolones, sulfonamides, tetracyclines, and trimethoprim. This study describes, for the first time, the detection and genomic profiling of high-risk lineages of E. coli ST410 and ST617 from surface water in Mexico. Additionally, our results underscore the role of surface water as a reservoir for critical pathogenic and MDR E. coli clones and the need for the surveillance and monitoring of aquatic environments via WGS from the One Health perspective.

Transient response of serpinA3 during cellular stress.

We demonstrated that serpinA3c/k relocates from the cytoplasm to the apical tubular membrane (ATM) in chronic kidney disease (CKD), suggesting its secretion in luminal space in pathophysiological contexts. Here, we studied serpinA3c/k expression and secretion under different stressful conditions in vitro and in vivo. HEK-293 cells were transfected with a FLAG-tagged serpinA3c/k clone and exposed to H2 O2 or starvation. Both stressors induced serpinA3c/k secretion but with a higher molecular weight. Glycanase treatment established that serpinA3c/k is glycosylated. Site-directed mutagenesis for each of the four glycosylation sites was performed. During cellular stress, serpinA3c/k secretion increased with each mutant except in the quadruple mutant. In rats and patients suffering acute kidney injury (AKI), an atypical urinary serpinA3c/k excretion (uSerpinA3c/k) was observed. In rats with AKI, the greater the induced kidney damage, the greater the uSerpinA3 c/k, together with relocation toward ATM. Our findings show that: (1) serpinA3c/k is glycosylated and secreted, (2) serpinA3c/k secretion increases during cellular stress, (3) its appearance in urine reveals a pathophysiological state, and (4) urinary serpinA3 excretion could become a potential biomarker for AKI.

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing.

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish variety cultured. Because aquaculture practices are susceptible to microbial contamination derived from anthropogenic sources, extensive antibiotic usage is needed, leading to aquaculture systems becoming an important source of antibiotic-resistant and pathogenic bacteria of clinical relevance such as Escherichia coli (E. coli). Here, the antimicrobial resistance, virulence, and mobilome features of a pathogenic E. coli strain, recovered from inland farmed Oreochromis spp., were elucidated through whole-genome sequencing (WGS) and in silico analysis. Antimicrobial susceptibility testing (AST) and WGS were performed. Furthermore, phylogenetic group, serotype, multilocus sequence typing (MLST), acquired antimicrobial resistance, virulence, plasmid, and prophage content were determined using diverse available web tools. The E. coli isolate only exhibited intermediate susceptibility to ampicillin and was characterized as ONT:H21-B1-ST40 strain by WGS-based typing. Although only a single antimicrobial resistance-related gene was detected [mdf(A)], several virulence-associated genes (VAGs) from the atypical enteropathogenic E. coli (aEPEC) pathotype were identified. Additionally, the cargo of plasmid replicons from large plasmid groups and 18 prophage-associated regions were detected. In conclusion, the WGS characterization of an aEPEC isolate, recovered from a fish farm in Sinaloa, Mexico, allows insights into its pathogenic potential and the possible human health risk of consuming raw aquacultural products. It is necessary to exploit next-generation sequencing (NGS) techniques for studying environmental microorganisms and to adopt a one health framework to learn how health issues originate.

Toward Understanding the Molecular Recognition of Fungal Chitin and Activation of the Plant Defense Mechanism in Horticultural Crops.

Large volumes of fruit and vegetable production are lost during postharvest handling due to attacks by necrotrophic fungi. One of the promising alternatives proposed for the control of postharvest diseases is the induction of natural defense responses, which can be activated by recognizing molecules present in pathogens, such as chitin. Chitin is one of the most important components of the fungal cell wall and is recognized through plant membrane receptors. These receptors belong to the receptor-like kinase (RLK) family, which possesses a transmembrane domain and/or receptor-like protein (RLP) that requires binding to another RLK receptor to recognize chitin. In addition, these receptors have extracellular LysM motifs that participate in the perception of chitin oligosaccharides. These receptors have been widely studied in Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa); however, it is not clear how the molecular recognition and plant defense mechanisms of chitin oligosaccharides occur in other plant species or fruits. This review includes recent findings on the molecular recognition of chitin oligosaccharides and how they activate defense mechanisms in plants. In addition, we highlight some of the current advances in chitin perception in horticultural crops.

The phosphoenolpyruvate-pyruvate-oxaloacetate node genes and enzymes in Streptomyces coelicolor M-145.

The phosphoenolpyruvate-pyruvate-oxaloacetate node is a major branch within the central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle. Phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malic enzymes, and pyruvate kinase, among others, are enzymes included in this node. We determined the mRNA levels and specific activity profiles of some of these genes and enzymes in Streptomyces coelicolor M-145. The results obtained in the presence of glucose demonstrated that all genes studied of the phosphoenolpyruvate-pyruvate-oxaloacetate node were expressed, although at different levels, with 10- to 100-fold differences. SCO3127 (phosphoenolpyruvate carboxylase gene) and SCO5261 (NADP+-dependent malic enzyme gene) showed the highest expression in the rapid growth phase, and the mRNA levels corresponding to SCO5896 (phosphoenolpyruvate-utilizing enzyme gene), and SCO0546 (pyruvate carboxylase gene) increased 5- to 10-fold towards the stationary phase. In casamino acids, in general mRNA levels of S. coelicolor were lower than in glucose, however, results showed greater mRNA expression of SCO4979 (PEP carboxykinase), SCO0208 (pyruvate phosphate dikinase gene), and SCO5261 (NADP+-dependent malic enzyme). These results suggest that PEP carboxylase (SCO3127) is an important enzyme during glucose catabolism and oxaloacetate replenishment. On the other hand, phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase, and NADP+-malic enzyme could have an important role in gluconeogenesis in S. coelicolor.

Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea.

BACKGROUND: Escherichia coli strains include both commensal and virulent clones distributed in different phylogenetic groups. Antimicrobial resistance is an increasingly serious public health threat at the global level and integrons are important mobile genetic elements involved in resistance dissemination. This paper aims to determine the phylogenetic groups and presence of class 1 (intl1) and 2 (intl2) integrons in E. coli clinical isolates from children with diarrhoea, and to associate these characteristics with their antimicrobial resistance. METHODS: Phylogeny and presence of integrons (intl1 and intl2) were analysed by PCR and amplicon sequencing in 70 E. coli isolates from children with and without diarrhoea (35 of each group) from Sinaloa, Mexico; these variables were analysed for correlation with the antimicrobial resistance profile of the isolates. RESULTS: The most frequent phylogroups were A (42.9%) and B2 (15.7%). The E. coli isolates from children with diarrhoea were distributed in all phylogroups; while strains from children without diarrhoea were absent from phylogroups C, E, and clade I. The 17.1% of the isolates carried integrons (15.7% intI1 and 1.4% intI2); 28.6% of the isolates from children with diarrhoea showed the class 1 integron. Strains of phylogroup A showed the highest frequency of integrons (33.3%). The association of multidrug resistance and the presence of integrons was identified in 58.3% of strains isolated from children with diarrhoea included in phylogroups A and B2. The sequence analysis of intl1 and intl2 showed silent point mutations and similarities with plasmids of some APEC and AIEC strains. CONCLUSION: Commensal E. coli strains are potential disseminators of antimicrobial resistance, and the improvement in the use of antimicrobials to treat childhood diarrhoea is essential for the control of such resistance.

Draft genome sequence of Escherichia coli M51-3: a multidrug-resistant strain assigned as ST131-H30 recovered from infant diarrheal infection in Mexico.

OBJECTIVES: In this study, we report the draft genome sequence of a multidrug-resistant (MDR)Escherichia coli strain recovered from stool sample of an outpatient infant girl with acute diarrheal infection in Mexico. METHODS: Antimicrobial susceptibility testing and PCR-based detection of diarrheagenic E. coli (DEC) were performed. In addition, genomic DNA from E. coli strain M51-3 was sequenced using Ion Torrent PGM platform with 200-bp chemistry and generated reads were de novo assembled using SPAdes v3.11. The draft genome was annotated and analyzed regarding multilocus sequence typing (MLST), serotyping, fimH typing, plasmid replicons, acquired antimicrobial resistance and virulence genes using web tools available at the Center for Genomic Epidemiology. RESULTS: A draft genome comprising 5 088 545 bp in length and 5308 protein-coding sequences was generated. In silico typification revealed that E. coli strain M51-3 belongs to ST131-O25:H4-H30 pandemic subclone. Several genes associated with resistance to ß-lactams [blaTEM-1B], aminoglycosides [aph(3'')-Ib, aadA5, aph(6)-Id and aac(3)-IId], sulfonamides [sul1 and sul2], trimethoprim [dfrA17], and tetracycline [tet(A)] were identified. Besides, point mutations in gyrA, parC, and parE genes were detected. Interestingly, the enterotoxin-coding virulence gene senB was evidenced. CONCLUSIONS: To our knowledge, this is the first draft genome of an E. coli ST131-O25:H4-H30 strain recovered from infant diarrheal stool sample in Mexico. The genome sequence of E. coli M51-3 presented here will be helpful to understand the genomic diversity of this highly virulent and MDR successfully pandemic bacterial pathogen.

Whole-genome sequencing of Staphylococcus aureus L401, a mecA-negative community-associated methicillin-resistant strain isolated from a healthy carrier.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen of great concern owing to its antimicrobial resistance and virulence properties. Here we report the first draft genome sequence of a mecA-negative community-associated MRSA strain isolated from a healthy young Mexican paediatric carrier in order to reveal the genomic structure underlying the multidrug-resistant phenotype and to discover the virulence properties of this strain. METHODS: The draft genome sequence of S. aureus L401 was obtained using an Ion Torrent™ PGM platform. De novo assembled contigs were annotated, and antimicrobial resistance genes and virulence factors were identified using ResFinder and VirulenceFinder, respectively. In addition, a mutational survey of native pbp, gdpP and yjbH genes was performed. In silico multilocus sequence typing (MLST) and spa typing were also performed. RESULTS: S. aureus L401 has a genome size of 2 831 587 bp with 2799 protein-coding sequences. Various antimicrobial resistance genes conferring resistance to aminoglycosides, ß-lactams, fluoroquinolones and macrolide-lincosamide-streptogramin B antimicrobials were found. Although both mecA and staphylococcal cassette chromosome mec (SCCmec) elements were absent, a missense mutation in PBP3 was identified. Moreover, genes encoding exfoliative toxin A, γ- and ß-haemolysin, and several enterotoxins were also identified. S. aureus L401 belongs to ST109 and spa type t209. CONCLUSION: The availability of this genome will allow an insight into S. aureus resistance and virulence determinants as well as its epidemiology, lineage, evolution and genomic features involved in the paediatric commensal carriage.

Tricarboxylic acid cycle without malate dehydrogenase in Streptomyces coelicolor M-145.

The oxidation of malate to oxaloacetate is catalysed only by a nicotinamide adenine dinucleotide-dependent malate dehydrogenase encoded by SCO4827 in Streptomyces coelicolor. A mutant lacking the malate dehydrogenase gene was isolated and no enzymatic activity was detected. As expected, the ∆mdh mutant was unable to grow on malate as the sole carbon source. However, the mutant grew less in minimal medium with glucose and there was a delay of 36 h. The same behaviour was observed when the mutant was grown on minimal medium with casamino acids or glycerol. For unknown reasons, the mutant was not able to grow in YEME medium with glucose. The deficiency of malate dehydrogenase affected the expression of the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes, decreasing the expression of both genes by approximately two- to threefold.

Draft Genome Sequence of Escherichia coli Strain M15-4, a Typical Enteropathogenic E. coli Strain Isolated in Mexico.

We present here the first draft genome sequence of a typical enteropathogenic Escherichia coli serotype O55:H51 strain, M15-4, isolated from a 2-month-old infant girl with acute diarrhea. The study of this Mexican isolate will provide insights to the virulence and drug resistance traits involved in its pathogenic potential.

Draft Genome Sequence of a Mexican Community-Associated Methicillin-Resistant Staphylococcus epidermidis Strain.

We report here the first draft genome sequence of a Mexican communitarian methicillin-resistant Staphylococcus epidermidis (MRSE) strain whose genome harbors a wide variety of resistance determinants. The availability of this genome will allow the study of antibiotic resistance in Mexican staphylococci from a genomic perspective.

Human MMAA induces the release of inactive cofactor and restores methylmalonyl-CoA mutase activity through their complex formation.

Human mitochondrial methylmalonyl-CoA mutase (hMCM) is an isomerase that converts methylmalonyl-CoA to succinyl-CoA, a crucial step for the incorporation of some compounds derived from the diet into the central metabolism. hMCM employs highly reactive radicals from its cofactor (adenosylcobalamin, AdoCbl) to perform its reaction. Our previous work demonstrated that hMCM loses activity during catalysis and that the interaction with human MMAA (hMMAA), a GTPase protein, avoided this loss or restored hMCM activity. Even so, the mechanism by which hMMAA exerted these chaperone functions has not been described. In this work report that the formation and accumulation of OH2Cbl, the oxidized form of the AdoCbl cofactor formed during catalysis, is the cause of hMCM inactivation. Additionally, we demonstrate that the complex formation of hMCM/hMMAA decreases the rate of oxidized cofactor formation, protecting the hMCM enzyme. Moreover, an inactive model of hMCM was used to demonstrate that hMMAA is able to remove the damaged cofactor through GTP hydrolysis. Additionally, a modification in the kinetic parameters of hMCM in presence of hMMAA was observed, and for the first time, the in vivo localization of hMMAA and its colocalization with hMCM in human fibroblasts mitochondria were demonstrated.

High prevalence of multidrug-resistant Escherichia coli isolates from children with and without diarrhoea and their susceptibility to the antibacterial activity of extracts/fractions of fruits native to Mexico.

PURPOSE: This paper aims to evaluate the antimicrobial resistance of Esherichia coli isolates from children under 5 years old, with and without diarrhoea, who were hospital outpatients in Culiacan, Sinaloa, Mexico. It also looks at the antimicrobial activity of fruit extracts against selected multidrug-resistant (MDR) E. coli strains. METHODOLOGY: A total of 205 E. coli isolates from stool samples were collected from 94 children under 5 years old who were outpatients from two hospitals in the city of Culiacan, Sinaloa, Mexico, during the autumn/winter of 2003/04; their resistance profiles to 19 commercial antimicrobials were investigated using the Kirby-Bauer method. The antibacterial activities of extracts/fractions of fruits (i.e. uvalama, Vitex mollis; ayale, Crescentia alata; and arrayan, Psidium sartorianum) were evaluated using the broth microdilution method. RESULTS: All E. coli isolates were susceptible to amikacin, nitrofurantoin and meropenem, and approximately 96 % were resistant to at least one antimicrobial, especially carbenicillin (93.2 %), cefuroxime sodium (53.7 %), ampicillin (40 %) and trimethoprim/sulfamethoxazole (35.1 %). Likewise, the frequency of MDR strains (44.9 %) was high, and no significant association with diarrhoea symptoms was found. Remarkably, all fruit extracts/fractions showed antibacterial activity against some, but not all, MDR isolates. The lowest minimal inhibitory concentration values were for the hexane fraction of arrayan (0.25 mg ml-1). CONCLUSION: A high number of antimicrobial-resistant E. coli (especially to ß-lactams and sulfonamides) and MDR isolates were detected in children under 5 years old, irrespective of diarrhoea symptoms; this is novel information for Culiacan, Sinaloa, Mexico. Moreover, our results showed that the studied fruit extracts/fractions are potential alternative or complementary treatments for MDR E. coli strains.

Giardia duodenalis genotypes among schoolchildren and their families and pets in urban and rural areas of Sinaloa, Mexico.

INTRODUCTION: Giardiasis is a human health concern worldwide, especially among schoolchildren. Giardia duodenalis genotypes A and B are infective to humans, but their zoonotic potential remains controversial. In Mexico, the most prevalent genotype is A, but B was also detected in southeastern Mexico. In Sinaloa state, northwestern Mexico, giardiasis is highly prevalent, but Giardia genotypes have been poorly studied. METHODOLOGY: This study aimed to investigate the distribution and clinical-epidemiological correlation of G. duodenalis genotypes in schoolchildren and their families and pets in urban and rural areas of Sinaloa state, Mexico. RESULTS: Among 395 schoolchildren (274 urban, 121 rural), 76 (49 urban, 27 rural) were infected with G. duodenalis. In total, 22 families (15 urban, 7 rural) of infected schoolchildren, consisting of 60 family members (41 urban, 19 rural) and 21 pet dogs (15 urban, 6 rural) were examined; 10 family members (5 urban, 5 rural) and 5 pet dogs (3 urban, 2 rural) of 10 families (6 urban, 4 rural) were infected. After PCR-RFLP analyses of vsp417 and gdh genes, genotype prevalence among infected urban schoolchildren was 79.5% AI, 12.8% AII, and 7.7% mixed AI+B. However, only AI genotype was found in family members and pets. In the rural area, only the AI genotype was detected. Genotypes were not correlated with clinical manifestations. CONCLUSIONS: This paper shows the presence of B genotype in northwestern Mexico for the first time. Detection of AI genotype in dogs suggested the possible role of dogs as the reservoir for human giardiasis in Sinaloa, Mexico.

Heat shock protein 72 (Hsp72) specific induction and temporal stability in urine samples as a reliable biomarker of acute kidney injury (AKI).

We demonstrated that urinary heat shock protein of 72 KDa (Hsp72) is a sensitive biomarker for the early detection of acute kidney injury (AKI). However, whether Hsp72 induction during an AKI episode is kidney-specific is unknown, as well as, the degree of Hsp72 stability in urine samples. In rats that underwent bilateral renal ischemia and reperfusion (I/R), Hsp72 levels were evaluated in several tissues and in collected urines under different storage and temperature conditions, as well as in variable numbers of freeze-thaw cycles. The effect of room temperature and five freeze-thaw cycles on urinary Hsp72 levels was also evaluated in urine samples from AKI patients. We found that Hsp72 increased exclusively in the renal cortex of I/R group, emphasizing its performance as an AKI biomarker. Urinary-Hsp72 remained constant at room temperature (48 h), during 9 months of storage and was not affected by five freeze/thaw cycles.