NHE Gene Research Articles

Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives

In this study levels and types of microbial contaminants were investigated in 88 different plant-based ingredients including many that are used to manufacture dairy alternatives. Studied ingredients encompassed samples of pulses (pea, faba bean, chickpea, and mung bean), cereals/pseudocereals (oat, rice, amaranth and quinoa) and drupes (coconut, almond and cashew). The microbial analysis included: i) total viable count (TVC), ii) total aerobic mesophilic spore count (TMS), iii) heat resistant aerobic thermophilic spore count (HRTS), iv) anaerobic sulfite reducing Clostridium spore count (SRCS), and v) Bacillus cereus spore count (BCES). Microorganisms isolated from the counting plates with the highest sample dilutions were identified using 16S rRNA and MALDI-TOF MS analyses.Many of the investigated ingredients showed a high proportion of spores as part of their total aerobic mesophilic counts. In 63 % of the samples, the difference between TVC and TMS counts was 1 Log10 unit or less. This was particularly the case for the majority of pea isolates and concentrates, faba bean isolates, oat kernels and flakes, and for single samples of chickpea isolate, almond, amaranth, rice, quinoa, and coconut flours. Concentrations of TVC ranged between <1.0 and 5.3 Log10 CFU/g in different samples, and TMS varied between <1.0 and 4.1 Log10 CFU/g. Levels of HTRS, BCES and SRCS were generally low, typically around or below the LOD of 1.0 Log10 CFU/g.In total, 845 individual bacterial colonies were isolated belonging to 33 different genera. Bacillus licheniformis and B. cereus group strains were most frequently detected among Bacillus isolates, and these species originated primarily from pea and oat samples. Geobacillus stearothermophilus was the main species encountered as part of the HRTS. Among the Clostridium isolates, Clostridum sporogenes/tepidum were predominant species, which were mostly found in pea and almond samples. Strains with potential to cause foodborne infection or intoxication were typed using the PCR-based method for toxin genes detection. In the B. cereus group, 9 % of isolates contained the ces gene, 28 % contained hbl, 42 % cytK, and 69 % were positive for the nhe gene. Absence of the boNT-A and -B genes was confirmed for all isolated C. sporogenes/tepidum strains. Nearly all (98 %) B. licheniformis isolates were positive for the lchAA gene.Insight into the occurrence of microbial contaminants in plant-based ingredients, combined with knowledge of their key inactivation and growth characteristics, can be used for the microbial risk assessment and effective design of plant-based food processing conditions and formulations to ensure food safety and prevent spoilage.

Isolation and identification of a pathogenic strain of Bacillus cereus from bovine and establishment of a rapid detection method for RPA-LF

In order to identify the cause of death of a cattle that suddenly died on a cattle farm in Tongliao City, Inner Mongolia Autonomous Region, we isolated a strain of bacteria from the spleen and identified the pathogen. The study demonstrated that the isolated strain is Bacillus cereus. The isolates were highly pathogenic and contained toxin genes, such as hbl, nhe, and entFM, in addition to the groEL housekeeping gene. The isolated strain has been named TL strain (16S rDNA sequence number: OP028902, whole genome sequence number: CP129005). Isolates were deposited at China General Microbiological Culture Collection Center (CGMCC No.17626). In this study, we also developed a rapid recombinase polymerase amplification-based lateral flow (RPA-LF) for pathogenic Bacillus cereus. The virulence gene (nhe gene) was used as the target sequence to design RPA-LF assay primers and probes. The RPA reaction is fast (results can be observed in about 15 min), the results can be judged by the naked eye, and the specificity of the RPA-LF method is good. The RPA-LF method detects pathogenic Bacillus cereus in food, which reduces the risk of harm to human health. The RPA-LF assay provides a reference for the rapid detection of pathogenic Bacillus cereus.

Genome−wide expression profile analysis of the NHE and NKA gene family in Rachycentron canadum (Linnaeus, 1766) and its response to salinity adaptation

Genome−wide expression profile analysis of the NHE and NKA gene family in Rachycentron canadum (Linnaeus, 1766) and its response to salinity adaptation

Microbiological safety and quality survey of some dairy confectioneries with cream filling and topping.

Dairy confectionaries are recently categorized as an important part of different consumers' diets with increasing demand for cream-based cakes "Tourta" and gateau which are used as celebrating food for almost all occasions and celebrations. The study was designed for evaluating general quality and safety of such products. 100 cream-based (for topping and filing) samples; 50 cakes "Tourta" and 50 gateau, were purchased separately from several pastry shops covering international, national, and local brands in Great Cairo, Egypt, and subjected to microbiological analysis. Results showed that international brands were the best for gateau samples while national brands were the best quality for cake samples. Regardless of the brand, the general hygienic quality of the cake product "Tourta" was lower on an average total colony, coliforms, yeast, and mold counts as compared with the gateau product. Although coliforms were found in 100% of the examined samples with the highest mean value of 33 × 102 ± 7.0 × 102 CFU/g in local gateau samples, Escherichia coli and Salmonella spp. could not be detected in these samples. Staphylococcus aureus was isolated from 8% of the total examined samples with the highest incidence of (22.2%) and a mean count of 38 × 10 ± 6.0 × 10 CFU/g in local brand gateau samples. Bacillus cereus was extensively isolated with the highest incidence in cake samples (32%) and mean counts higher than 103 in all of the examined samples. Bacillus cereus strains have harbored more than one toxigenic gene. In this aspect, nhe gene was the most predominant one as it was detected in 100% of the examined isolates, followed by cytK gene in 80%, whilehbl and ces genes could not be found. According to the Egyptian Standard specifications for cake (ES: 4037/2020), a higher acceptability degree was reported for the international brand gateau and cake "Tourta" samples. The incidence of using any preservative as an inhibitory substance was also analyzed generally using the Bacillus subtilis disk assay technique, but all samples were negative using this technique indicating the need for a more advanced technique for its detection such as using high-performance liquid chromatography. Finally, it was concluded that; more attention is needed to cream-based cakes' quality and safety, with essential modification required in the standards of cakes in Egypt.

Genome-wide identification of the NHE gene family in Coilia nasus and its response to salinity challenge and ammonia stress

BackgroundIn aquatic environments, pH, salinity, and ammonia concentration are extremely important for aquatic animals. NHE is a two-way ion exchange carrier protein, which can transport Na+ into cells and exchange out H+, and also plays key roles in regulating intracellular pH, osmotic pressure, and ammonia concentration.ResultsIn the present study, ten NHEs, the entire NHE gene family, were identified from Coilia nasus genome and systemically analyzed via phylogenetic, structural, and synteny analysis. Different expression patterns of C. nasus NHEs in multiple tissues indicated that expression profiles of NHE genes displayed tissue-specific. Expression patterns of C. nasus NHEs were related to ammonia excretion during multiple embryonic development stages. To explore the potential functions on salinity challenge and ammonia stress, expression levels of ten NHEs were detected in C. nasus gills under hypotonic stress, hypertonic stress, and ammonia stress. Expression levels of all NHEs were upregulated during hypotonic stress, while they were downregulated during hypertonic stress. NHE2 and NHE3 displayed higher expression levels in C. nasus larvae and juvenile gills under ammonia stress.ConclusionsOur study revealed that NHE genes played distinct roles in embryonic development, salinity stress, and ammonia exposure. Syntenic analysis showed significant difference between stenohaline fish and euryhaline fishes. Our findings will provide insight into effects of C. nasus NHE gene family on ion transport and ammonia tolerance and be beneficial for healthy aquaculture of C. nasus.

Aerobic spore-forming bacteria in powdered infant formula: Enumeration, identification by MALDI-TOF mass spectrometry (MS), presence of toxin genes and rpoB gene typing

This study aimed to assess the counts and biodiversity characterization of aerobic sporeforming bacteria (ASB) in powdered infant formula (PIF). Fifty-four (n = 54) samples of PIF of three brands were analyzed for mesophilic aerobic bacteria, and ASB counts. ASB isolated from PIF were assessed for their ability to produce spoilage enzymes and hemolytic activity and further identified by MALDI-TOF mass spectrometry. Then, the isolates were subjected to rpoB gene typing and assessment of bceT, entFM, nhe (A, B, C), and hbl (A, B, C) toxin genes. The main species isolated were B. licheniformis (54%), followed by B. cereus (33%) and B. subtilis (5%). The ASB counts ranged from 1 to 4 log CFU/g, and the mean was 2.9 log CFU/g for mesophilic aerobic sporeforming bacteria (MSC) and 2.5 log CFU/g for thermophilic aerobic sporeforming bacteria (TSC). Most PIF samples presented MSC and TSC counts between 2 and 3 log CFU/g. A total of 13%, 50%, and 37% of the samples presented MSC counts from <2 log CFU/g, between 2 and 3 log CFU/g and between 3 and 4 log CFU/g, respectively. Among the ASB isolates, 97% had protease, 84% hydrolyzed starch, 66% had hemolytic activity, and 61% had lecithinase activity. A total of 44 out of 120 isolates harbored at least one toxin gene; 56% for B. cereus, 34% for B licheniformis, and less than 5% for B. subtilis, B pumilus, and L. sphaericus. All B. cereus isolates harbored the nhe gene, 60% entFM, 44% cytK, 32% bceT, and 28% hbl genes. Besides, 17% of B. licheniformis harbored nhe. A small proportion of B. subtilis, B. pumilus, and L. sphaericus carried toxin genes. The rpoB based phylogenetic tree provided high resolution among Bacillus species. The findings of this study provide insights into the phenotypic and genotypic biodiversity of Bacillus present in PIF.

Comprehensive Study about the Identification of Bacillus Cereus Isolated from Chicken Farms

Bacillus cereus is one of the opportunistic foodborne bacteria producing food poisoning producing severe economic loss and representing a high risk to the human. The contamination of poultry feed with Bacillus cereus produces gastric damage and facilitates other bacterial infections. For these reasons, fresh fecal matters were collected from poultry farms located in 3 Egyptian Governorates (Giza, Gharbia, and Dakahlia) to isolate and identify Bacillus cereus, determine their susceptibility to different antimicrobial agents, and examine their virulence and resistance factors. Eleven B. Cereus out of 50 examined samples was isolated with a percentage of (11/50, 22%) and subjected to confirmation using the VITEK2 instrument. Disc diffusion tests showed that all isolates were resistant to trimethoprim-sulfamethoxazole, imipenem, cefotaxime, ampicillin, amoxicillin/clavulanic acid, and penicillin G and sensitive to chloramphenicol, gentamicin, and ciprofloxacin. The molecular characterizations of virulence and resistance factors revealed the detection of targeting the groEL gene in all isolates (100%). The toxin gene profiling showed the existence of the nhe gene (90.9%) and the hbl genes (18.18%). The ces and cytk genes were not detected in the examined isolates. Two antimicrobial resistance genes were detected in the examined isolates with (33.33%) for the tetA gene and (45.45%) for bla gene. Therefore, it was concluded that the isolation of B. cereus carrying genes responsible for antimicrobial resistance and toxins production in broiler farms, represents major threats for human health and poultry industry. So that control programs of biosecurity should be implemented in poultry farms to mitigate the losses of B. cereus infection.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Characterization of Bacillus cereus Group Isolates From Human Bacteremia by Whole-Genome Sequencing.

Members of the Bacillus cereus group are spore-forming organisms commonly associated with food poisoning and intestinal infections. Moreover, some strains of the group (i.e., B. cereus sensu stricto and Bacillus thuringiensis) can cause bacteremia in humans, mainly in immunocompromised individuals. Here we performed the genetic characterization of 17 human clinical strains belonging to B. cereus group isolated from blood culture. The whole-genome sequencing (WGS) revealed that the isolates were closely related to B. cereus sensu stricto and B. thuringiensis–type strain. Multilocus sequence typing analysis performed on the draft genome revealed the genetic diversity of our isolates, which were assigned to different sequence types. Based on panC nucleotide sequence, the isolates were grouped in the phylogenetic groups III and IV. The NHE, cer, and inhA gene cluster, entA, entFM, plcA, and plcB, were the most commonly detected virulence genes. Although we did not assess the ability to generate biofilm by phenotypic tests, we verified the prevalence of biofilm associated genes using an in silico approach. A high prevalence of pur gene cluster, xerC, clpY, codY, tasA, sipW, sinI, and sigB genes, was found. Genes related to the resistance to penicillin, trimethoprim, and ceftriaxone were identified in most of the isolates. Intriguingly, the majority of these virulence and AMR genes appeared to be evenly distributed among B. cereus s.s. isolates, as well as closely related to B. thuringiensis isolates. We showed the WGS represents a good approach to rapidly characterize B. cereus group strains, being able to give useful information about genetic epidemiology, the presence of virulence and antimicrobial genes, and finally about the potential hazard related to this underestimated risk.

Prevalence of Virulence Determinants among Bacillus cereus Isolated from Milk Products with Potential Public Health Concern.

Bacillus cereus is described as one of the public health pathogen causing severe food poisoning outbreaks worldwide. Accurate surveillance about B. cereus in Egypt is scanty. For this reason, the current study was conducted to determine the prevalence of B. cereus and its virulence genes among milk powder and Ras-cheese products. Two hundred samples (130 and 70) from milk powder and Ras-cheese, respectively were aseptically collected and cultured onto specific media. The obtained isolates were subjected to mPCR for screening of virulence genes (nhe, cytK, pc-plc, hblD, hbI and ces) among of B. cereus isolates that obtained from milk powder and Ras-cheese. The result revealed that B. cereus was recovered with 6.9 and 8.5% from milk powder and Ras-cheese, respectively. The nhe gene was detected and dominated in all isolates 100% from both products. In milk powder, pc-plc was the most prevalent gene (100%). However, cytK, hblD, hbl and ces genes were prevalent with 55.5, 33.3, 33.3 and 22.2%, respectively. Regarding to Ras-cheese, the prevalence of cytK was (83.33%) while each of hbI, hblD, pc-plc and ces genes were recovered in 50% of tested isolates. This result provided an important epidemiological view about the contamination rate and the most prevalent virulence genes of B. cereus in milk products in Egypt.

Bacillus cereus: public health burden associated with ready-to-eat foods in Himachal Pradesh, India.

The study determined incidence, enterotoxigenecity and antimicrobial susceptibility profiles of Bacillus cereus isolated from ready-to-eat (RTE) milk products (n = 80), RTE meat products (n = 40), beverages (n = 40) and water samples (n = 60, from food preparing and serving outlets/restaurants) collected from eight different tourist places of Himachal Pradesh. 11.4% (25/220) samples were contaminated with Bacillus and isolates were identified as B. cereus (76.0%, n = 19), B. alvei (12.0%, n = 3), B. polymyxa (8.0%, n = 2) and B. firmus (4.0%, n = 1) by conventional and molecular methods. B. cereus incidence was highest in cheese based foods (25.0%) followed by vegetable soups (16.7%), khoa based foods (14.0%), milk based beverages (10.5%), paneer based foods (8.6%), cream based foods (8.3%) and water (8.3%) samples. Multiplex polymerase chain reaction detected enterotoxigenic genes only in B. cereus isolates. nhe complex (encoding non-haemolytic enterotoxins, ABC) genes were detected only in B. cereus isolates. 57.6% (11/19), 36.8% (7/19) and 5.3% (1/19) harboured all three (nheA, nheB, nheC), two (nheB, nheC) and one (nheC) nhe gene, respectively. Among hbl complex genes (encoding haemolytic enterotoxins CAD), only hblC (36.8%, 7/19)was detected. Incidence B. cereus cytK (encoding cytotoxin enterotoxin) was 52.6% (10/19). Each B. cereus isolate harboured two or more enterotoxigenic genes. Seven isolates had at least one gene from haemolytic and non-haemolytic complexes along with cytK. High levels (> 50%) of antimicrobial resistance were recorded for penicillin, amoxicillin, ampicillin cefixime and ceftazidine in tested B. cereus isolates. Two isolates were identified as multidrug resistant isolates with resistance to ≥ 3 antibiotic classes.

Enterotoxigenic Profiles of Bacillus Cereus Isolated from Manufacturing Steps of Traditional Soft Cheese Plant

Microbial contamination of soft cheese may originate from various sources such as contaminated equipment or from the workers themselves during cheese production. The study was aimed to characterize Bacillus cereus using culture-based and PCR techniques among 285 samples along Damietta and Kareish cheese producing line in a small cheese plant in Alexandria governorate including raw milk, cheese, whey, rennet, water, and swabs from all contacts surfaces. Overall of 10.90 % and 10 % among raw milk, cheese, whey, rennet, water, and swabs from all contacts surfaces were positive for Bacillus species in Damietta and Kareish cheese producing line. The incidence of Bacillus cereus in receiving raw milk, curd, whey, fresh cheese samples were 6.66, 13.33 and 13.33%, respectively for traditional Damietta cheese plant. Bacillus cereus is found in only 6.66 % in receiving raw milk, pasteurized milk and fresh cheese samples for traditional Kareish cheese plant. Hbl, Nhe and Cytk genes were detected in Bacillus cereus isolated from curd and Damietta cheese. Nhe gene was detected in Bacillus cereus isolated from raw milk, pasteurized milk and Kareish cheese While, Ces gene was detected only in Bacillus cereus isolated from fresh Kareish cheese samples. The prevalence of Eneterotoxigenic virulent B. cereus poses public health threats to consumers. HACCP should be implemented for all steps of manufacture in order to produce a safe and good quality dairy product.

Genome-wide identification of the Na+/H+ exchanger gene family in Lateolabrax maculatus and its involvement in salinity regulation

Na+/H+ exchangers (NHEs) are one of the major groups of transmembrane proteins that play crucial roles in pH homeostasis, cell volume regulation and Na+ transport in animals. In our study, twelve NHEs were identified from transcriptomic and genomic databases of Lateolabrax maculatus. The evolutionary footprint of each NHE gene was revealed via the analysis of phylogenetic tree, copy numbers, exon-intron structures and motif compositions. NHEs harbored a high proportion of α-helices (54.7% to 67.0%) and a low proportion of β-sheets (1.3%) and contained 9–13 transmembrane helices (TM). Results of tissue distribution detection revealed that L. maculatus NHE genes exhibited different expression profiles in a tissue-specific manner under normal physiological conditions. In the main osmoregulatory organ, gill, NHE2c and NHE3 showed significant higher expression comparing with other L. maculatus NHE genes. To gain insight into the potential function of L. maculatus NHE genes in response to salinity changes, we evaluated their expression variation after different salinity treatment (0 ppt, 12 ppt, 30 ppt, 45 ppt). During acute salinity stress experiment, L. maculatus NHE genes were regulated in a gene-specific and time-dependent manner. Most NHE genes were upregulated to different extent by low salinity (0 ppt) and exhibited the highest expression value in gills at 6 h, while NHE2c and NHE3 were the most strongly induced genes, suggesting they may play crucial roles in salinity and osmotic regulation. In addition, the expressions of several NHE genes were regulated by isotonic or high salinity treatments, indicating their potential involvements in response to salinity challenge. Our findings in this study provide a foundation for future studies about NHE gene deciphering stress physiology correlated to salinity and osmotic regulation in teleosts.

Enterotoxigenic structures of Bacillus cereus strains isolated from ice creams

AbstractThis study was conducted to investigate the presence of Bacillus cereus in ice cream samples and to identify associated toxin genes by mPCR. 125 ice cream samples were used as material. A total of 38 samples were found to be positive for B. cereus. It was found that 31.9% of the isolates had three enterotoxic HBL complex encoding genes, 10.6% had two hbl genes and 6.3% contained one hbl gene. On the other hand, 15.9% of the isolates contained three NHE complex encoding genes, 31.9% had two nhe genes and 20.2% contained one nhe gene. Also 7.4% of isolates were found to contain both NHE and HBL complexes while ctyK1 was not detected from any isolate. The presence of B. cereus and their enterotoxigenic genes in ice creams may be a potential risk for public health.Practical applicationsThe presence of the B.cereus in high numbers and the toxins in foods pose a potential risk in terms of health and food spoilage. In food poisoning cases, hbl, nhe, cytK, and the effect of emetic toxin are especially notable. The resistance of spores against pasteurization and psychrotolerant feature enable the explanation of the existence of B. cereus in ice‐cream.

Toxin genes and cytotoxicity levels detected in Bacillus cereus isolates collected from cooked food products delivered by Swiss Army catering facilities.

Heated food is known to be often contaminated with B. cereus, leading to cases of diarrhoeal or emetic diseases. Battalion kitchens or army catering facilities present a food safety risk, as temperature abuse and long storage time can result in serious public health problems affecting a high number of served people. In contrast to civil catering facilities, no microbiological monitoring systems are currently implemented in Swiss military kitchens. In this study toxin gene profiles and cytotoxicity levels of 21 isolates of B. cereus originating from six different food categories were determined. Nearly all isolates (95%) harbored the nhe gene, whereas no hbl could be detected. Seven isolates displayed the cytK2 gene and one cereulide-producer was isolated out of vegetables. While most isolates displayed low cytotoxicity, highly cytotoxic strains were detected, with three isolates even exceeding the cytotoxicity level of the reference strain for high-level toxin production, underpinning that cytotoxicity cannot be deduced only from presence or absence of toxin genes. These findings further underline the importance of rapid cooling of foods or maintenance over 65°C before serving. This is especially important in mass catering facilities, such as military kitchens, in which food is often prepared a long time in advance.

Sütlü Tatlılardan İzole Edilen Bacillus cereus Suşlarının Enterotoksijenik Gen Profillerinin Multipleks PCR ile Belirlenmesi

Summary The aim of this study was to investigate the presence of Bacillus cereus and to detect enterotoxigenic genes in dairy dessert samples utilising a multiplex PCR technique. A total of 100 samples, 25 keskul, 12 tavuk gogsu, 12 kazandibi, 30 supangle, 4 profiteroles and 17 sutlac were analysed. B. cereus contamination was found in 7 of 100 (7.0%) samples including 3 (25%) tavuk gogsu, 2 (6.6%) supangle, 1 (4%) keskul and 1 (5.8%) sutlac. The average number of B. cereus was between 2.0×10 1 - 5.0×10 2 cfu/g in these dairy dessert samples. A total of 20 isolates were collected from the 7 positive samples. The results indicated that 30% (6/20) of B. cereus isolates contain three enterotoxic HBL complex encoding genes hblA, hblC and hblD, whereas 70% (14/20) had no hbl genes. In addition, all three enterotoxic NHE complex encoding genes, nheA, nheB and nheC, were detected in 40% (8/20), two nhe genes (nheA and nheB) were found in 45% (9/20) and one nhe gene (two nheA and one nheB) was found in 15% (3/20) of the isolates. The ctyK1 gene was not detected in any sample. The presence of B. cereus and their enterotoxigenic genes in dairy desserts may be a potential risk for public health.

Bacillus cereus infection outbreak in captive psittacines

This study reports an uncommon epizootic outbreak of Bacillus cereus that caused the sudden death of 12 psittacines belonging to the species Anodorhynchus hyacinthinus (1 individual), Diopsittaca nobilis (1 individual), Ara severa (1 individual) and Ara ararauna (9 individuals) in a Brazilian zoo. Post-mortem examination of the animals reveled extensive areas of lung hemorrhage, hepatic congestion, hemorrhagic enteritis and cardiac congestion. Histopathological examination of the organs showed the presence of multiple foci of vegetative cells of Gram-positive bacilli associated with discrete and moderate mononuclear inflammatory cell infiltrate. Seventeen B. cereus strains isolated from blood and sterile organs of nine A. ararauna were analyzed in order to investigate the genetic diversity (assessed by Rep-PCR) and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. Amplification of genomic DNA by Rep-PCR of B. cereus strains generated two closely related profiles (Rep-PCR types A and B) with three bands of difference. All strains were classified as belonging to the toxigenic profile I which contained HBL and NHE gene complexes, entFM and cytK genes. Altogether, microbiological and histopathological findings and the evidence provided by the success of the antibiotic prophylaxis, corroborate that B. cereus was the causative agent of the infection that killed the birds.

Organellar (Na+, K+)/H+ exchanger NHE7 regulates cell adhesion, invasion and anchorage-independent growth of breast cancer MDA-MB-231 cells

Na+/H+ exchangers (NHEs) are a group of secondary active antiporters that regulate cellular pH, cell volume and ion homeostasis. In humans, nine isoforms (NHE1-NHE9) were identified and characterized as functional NHEs. While a growing body of evidence indicates that NHE1 generates an acidic tumor environment and thereby contributes to tumor invasion, little is known about the role of other NHE isoforms in tumor progression. NHE7 is a unique member of the NHE gene family that dynamically shuttles between the trans-Golgi network, endosomes and the plasma membrane, and regulates the luminal pH of these organelles. Here we show that NHE7-overexpression in breast cancer MDA-MB-231 cells enhances cell overlay, cell-cell adhesion, invasion, anchorage-independent tumor growth and tumor formation invivo. In contrast, NHE1-overexpression enhances tumor invasion, but it has little effect on cell adhesion or anchorage-independent tumor growth. Pathological examinations of the tumor samples derived from NHE7-overexpressing cells showed a similar appearance to aggressive tumors. Together, these results suggest that NHE7 enhances tumor progression. This is the first report to show the involvement of an organellar NHE in oncogenic processes.

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na(+)/H(+) exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.

MOLECULAR PHYSIOLOGY OF INTESTINAL N+/H+ EXCHANGE

The sodium/hydrogen exchange (NHE) gene family plays an integral role in neutral sodium absorption in the mammalian intestine. The NHE gene family is comprised of nine members that are categorized by cellular localization (i.e., plasma membrane or intracellular). In the gastrointestinal (GI) tract of multiple species, there are resident plasma membrane isoforms including NHE1 (basolateral) and NHE2 (apical), recycling isoforms (NHE3), as well as intracellular isoforms (NHE6, 7, 9). NHE3 recycles between the endosomal compartment and the apical plasma membrane and functions in both locations. NHE3 regulation occurs during normal digestive processes and is often inhibited in diarrheal diseases. The C terminus of NHE3 binds multiple regulatory proteins to form large protein complexes that are involved in regulation of NHE3 trafficking to and from the plasma membrane, turnover number, and protein phosphorylation. NHE1 and NHE2 are not regulated by trafficking. NHE1 interacts with multiple regulatory proteins that affect phosphorylation; however, whether NHE1 exists in large multi-protein complexes is unknown. Although intestinal and colonic sodium absorption appear to involve at least NHE2 and NHE3, future studies are necessary to more accurately define their relative contributions to sodium absorption during human digestion and in pathophysiological conditions.