Retrospective study reveals the circulation of norovirus genotype GII.P21-GII.2 in Romania

Noroviruses are the leading cause of acute gastroenteritis, causing significant economic burden globally. Infection is self-limiting, occurring as sporadic cases or producing outbreaks associated with consumption of contaminated water or food. All age groups are affected and person to person transmission is frequent. Except a recent outbreak in Romania caused by the emergent genotype GII.P17-GII.17, few data regarding the circulation of noroviruses in our country are available. We retrospectively analyzed stool samples from acute gastroenteritis patients hospitalized in Romania between 2005 and 2008. Noroviruses were detected by RT-PCR and phylogenetic analysis was inferred from partial sequences spanning ORF1 and ORF2. Recombinant GII.P21-GII.2 isolates were found in two adult patients from a cluster of acute gastroenteritis in 2006. Molecular analysis based on partial genomic sequences indicated high degree of similarity between the two isolates and grouped them with cosmopolitan strains circulating in the same period of time. Along with the high rate of mutation, recombination is an important driving force in norovirus evolution. GII.P21 isolates, formerly known as GII.b recombinants, have been detected in Europe since 2000 and associated with sporadic cases and outbreaks of gastroenteritis worldwide. This is the first work describing norovirus GII.P21-GII.2 identified in Romania.

Method for the determination of ammonium in cigarette tobacco using ion chromatography.

Ammonia and other alkaline substances have been postulated to be important in cigarette design. The most significant potential contribution of ammonia is a possible interaction with the native, protonated nicotine in the smoke. Ammonia is more alkaline than nicotine and could facilitate a shift in the acid/base equilibrium where a fraction of the total nicotine converts to the more lipophilic, non-protonated form. This non-protonated, or free-base, form of nicotine absorbs more efficiently across membranes, resulting in more rapid delivery to the smoker's bloodstream. Ammonia and other potential ammonia sources, such as additives like diammonium phosphate, could influence the acid-base dynamics in cigarette smoke and ultimately the rate of nicotine delivery. To examine and characterize the ammonia content in modern cigarettes, we developed a fast, simple and reliable ion chromatography based method to measure extractable ammonia levels in cigarette filler. This approach has minimal sample preparation and short run times to achieve high sample throughput. We quantified ammonia levels in tobacco filler from 34 non-mentholated cigarette brands from 3 manufacturers to examine the ranges found across a convenience sampling of popular, commercially available domestic brands and present figures of analytical merit here. Ammonia levels ranged from approximately 0.9 to 2.4mg per gram of cigarette filler between brands and statistically significance differences were observed between brands and manufacturers. Our findings suggest that ammonia levels vary by brand and manufacturer; thus in domestic cigarettes ammonia could be considered a significant design feature because of the potential influence on smoke chemistry.

New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.

Phenotypic and molecular methods used for identification of oral streptococci and related microorganisms.

The oral cavity contains the greatest biodiversity, over 70 species being isolated from mouth mucosa, saliva, denture surfaces and/or dental-plaque. The oral streptococci, representing over 80% of the mouth micro flora, are able to synthesize glucosyl-transferases, enzymes involved in glucans production. Glucans are involved in production of an extracellular slime layer promoting adhesion and formation of a dental plaque biofilm. The 43 isolates studied obtained from partially and/or totally edentulous, were identified by VITEK system using gram-positive identification cards. Species-specific regions within the genes coding for glucosyl-transferases (gtf genes) were targeted for PCR identification of isolates. Sequencing of 16S rRNA was used as gold standard for strain confirmation. VITEK system identified a number of 11 strains as S. mitis/oralis, 12 strains as S. anginosus/gordonii, 12 strains as S. sanguinis/parasanguinis, 3 strains as S. salivarius, 3 strains as S. plurianimalium, 1 strain as S. cristatus and 1 strain as S. alactolyticus, respectively. The PCR system targeting gtf genes was able to identify S. oralis, S. salivarius and S. gordonii strains. Sequence of 16S rRNA discriminated among streptococci species and revealed 16 strains of Leuconostoc mesenteroides. Many studies are needed in order to select the most reliable phenotypic and genotypic methods in order to improve the identification algorithm for oral streptococci used by clinical laboratories. Their accurate identification is mandatory for better understanding their role in human infections.

A multiple typing approach is recommended for the laboratory-based surveillance of salmonellosis in Romania.

In Romania, Salmonella enterica serovar Typhimurium isolates are currently typed by antimicrobial resistance profiles and phage typing, as part of the national laboratory-based surveillance system of human enteric infections. The aim of the present study was to assess the added value of complementing this approach with molecular fingerprinting, namely pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeats analysis (MLVA). Thirty-six S. Typhimurium isolates received by the Reference Center for Human Salmonella Infections for confirmation and typing from the Microbiology Departments of three Public Health Authorities, were selected for this study. Phage typing revealed that 14 isolates (39%) were nontypeable (NT). Twenty-two isolates were assigned to 5 phage types: DT193 (11 isolates), U302 (7 isolates), DT116 (2 isolates), DT41 (1 isolate) and DT86 (1 isolate). Antimicrobial susceptibility testing showed that all the NT and DT116 isolates were multidrug resistant and extended-spectrum betalactamase producers. All the examined isolates were typeable when using the molecular approach. Both methods gave conclusive and comparable results, documenting the genetic relatedness and discriminating the outbreak isolates from sporadic cases. We conclude that in order to improve outbreak investigation and surveillance of salmonellosis in Romania, the current routine typing of Salmonella isolates should be complemented with at least one of these DNA fingerprinting methods.

[Bacterial enteric pathogens' resistance to fluoroquinolones and last generation cephalosporines]. / Rezistenta patogenilor enterici bacterieni la fluoroquinolone si cefalosporine de ultima generatie.

The increase of incidence of resistance to the antibiotics became the most worrisome subject within the clinical and research communities in the medical fields. Intrinsic resistance genetic mutations, horizontal transfer of mobile structures carrying genes coding for resistance to the antibiotics within the pan-microbial genome are representing the bacterial resistome which is bearing the genetic information regarding the defensive mechanisms developed by micro-organisms to protect themselves against antibiotics. Rice in the resistance of enteric bacteria, pathogens involved in a large number of human infections, to the cephalosporin of last generation and to the fluoroquinolones is a very actual subject in the medical area. Production of beta-lactamases with extended spectrum is the most important enzymatic defence system, developed by micro-organisms, consisting in the inactivation of beta-lactam antibiotics by destroying the beta-lactam ring. Enterobacteria are able to produce beta-lactamases of type TEM, SHV and/or CTX-M. Punctual mutations in nucleotide structure of bla genes, coding for beta-lactamases synthesis, are leading on production of a large diversity of enzymes with enlarged spectrum of activity (ESBL). At the beginning of 90's the first beta-lactamases resistance to clavulanic acid were detected and in our days more then 170 TEM, 120 SVH and 90 CTX-MESBLs are known. Escherichia coli strains are producing, firstly, TEM ESBLs, Klebsiella pneumoniae SHV ESBLs. and both are producing CTX-M type ESBLs, are resistant to the fluoroquinolones due to punctual mutations in nucleotide structure of gyr gene coding for gyrases production, enzymes involved in nucleic acids replication. Resistance to the antibiotics with extended activity is a public health threat due to their capacity of large spreading within bacterial population, when the coding structures are located on mobile genetic structures. The menace increase when genes coding for fluoroquinolones resistance (qnr) are identified on such of structures.

The seventh pandemic of cholera in Europe revisited by microbial genomics.

In 1970, the seventh pandemic of cholera (7 P) reached both Africa and Europe. Between 1970 and 2011, several European countries reported cholera outbreaks of a few to more than 2,000 cases. We report here a whole-genome analysis of 1,324 7 P V. cholerae El Tor (7 PET) isolates, including 172 from autochthonous sporadic or outbreak cholera cases occurring between 1970 and 2011 in Europe, providing insight into the spatial and temporal spread of this pathogen across Europe. In this work, we show that the 7 PET lineage was introduced at least eight times into two main regions: Eastern and Southern Europe. Greater recurrence of the disease was observed in Eastern Europe, where it persisted until 2011. It was introduced into this region from Southern Asia, often circulating regionally in the countries bordering the Black Sea, and in the Middle East before reaching Eastern Africa on several occasions. In Southern Europe, the disease was mostly seen in individual countries during the 1970s and was imported from North and West Africa, except in 1994, when cholera was imported into Albania and Italy from the Black Sea region. These results shed light on the geographic course of cholera during the seventh pandemic and highlight the role of humans in its global dissemination.

Role of deoxyribose catabolism in colonization of the murine intestine by pathogenic Escherichia coli strains.

We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by the deoK operon, is associated with the pathogenic potential of Escherichia coli strains. Carbohydrate metabolism is thought to provide the nutritional support required for E. coli to colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenic E. coli strains. Molecular and biochemical characterization of 1,221 E. coli clones from various collections showed this biochemical trait to be common in the E. coli species (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizing E. coli clones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenic E. coli were significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of the deoK operon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenic E. coli growth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.

Escherichia coli pathotypes associated with diarrhea in Romanian children younger than 5 years of age.

To document the association of pathogenic Escherichia coli with diarrhea in Romanian children, 250 E. coli fecal isolates from children under 5 years of age were PCR-screened for well-recognized virulence determinants, as well as for their phylogenetic background. The putative diarrheagenic E. coli (DEC) were investigated for susceptibility to various antibiotics. Overall, 61 E. coli isolates were classified as enteroaggregative E. coli (29 isolates), atypical enteropathogenic E. coli (22 isolates), enterotoxigenic E. coli (8 isolates), and verotoxin-producing E. coli (1 isolate), and one isolate was categorized as unconventional DEC. Only 8 of the PCR-positive isolates would have been assumed to be pathogenic based on their O antigenicity, which highlights the limited effectiveness of serotyping. More than a half (51%) of the pathogenic isolates expressed a multidrug-resistant phenotype, which raises concerns about the therapeutic pediatric approach. The DEC isolates were heterogeneous phylogenetically, deriving from all four major groups: A (31 isolates), B2 (14 isolates), B1 (10 isolates), and D (6 isolates), respectively. Thus, the phylogenetic descent was less significant than the virulence gene content. Our findings document the importance of DEC as a cause of childhood diarrhea in Romania, providing evidence that efforts should be made to estimate the burden of infections by etiology for a better medical approach.

Identification of plasmid-mediated quinolone resistance qnr-like genes in Romanian clinical isolates of Escherichia coli and Klebsiella pneumoniae.

A collection of putative ESBL-producing Escherichia coli (119 isolates) and Klebsiella pneumoniae (122 isolates) originating from extraintestinal human specimens was screened for qnrA, qnrB, and qnrS-like genes by PCR. Seven K. pneumoniae isolates, which were resistant to ciprofloxacin, were detected as carrying qnrA1-like genes, while one K. pneumoniae and one E. coli isolate were positive for qnrS-like determinant. The latter isolates were susceptible to ciprofloxacin. This is the first study identifying qnr-like genes in our area. Further studies are needed to document the contribution of the plasmid mediated quinolone resistance to the increase in bacterial resistance to fluoroquinolones in Romania.

Pulsed-field gel electrophoresis associated to phage typing improves the discrimination of epidemiologically unrelated Salmonella enterica serovar typhimurium isolates.

A combination of phage typing and pulsed-field gel electrophoresis (PFGE) of Xbal- and Blnl-digested chromosomal DNA has been used to study 18 epidemiologically unrelated human Salmonella enterica serovar Typhimurium isolates, which were collected during 2007 within a single Romanian county. Phage typing could assign only four of the isolates to three definitive phage types (DT41, DT86, and DT116), the rest being untypable by this classical method. PFGE analysis of the double enzyme-digested DNA, performed in an attempt to further discriminate the strains, allowed the typing of all the studied isolates. Xbal-digested genomic DNA segregated the isolates into 7 X-types and Blnl restriction differentiated them into 8 B-types. Our PFGE results documented the circulation of a rather homogeneous population of S. Typhimurium strains within the same county. As in the case of other human pathogens, epidemiological conclusions might be more accurate if based on both phenotypic and genotypic methods, therefore molecular typing should be added within the national laboratory-based surveillance of Salmonella infections.

Laboratory diagnosis of infectious diarrhoea syndrome; a three years study in two hospitals of infectious diseases.

Infectious diarrhoea is a syndrome caused by a variety of bacterial, viral and parasitic organisms which represents a major cause of morbidity and mortality all over the world. The wide diversity of etiological agents impairs the surveillance and the diagnosis and affects the correct treatment applied to reduce the long-term complications. Besides well known enteric pathogens such as Salmonella, Shigella and Yersinia, a high number of emergent and re-emergent aetiologies are now recognised to be at the origin of diarrhoea. The lack of a correct diagnostic algorithm and adequate methods of analyses leads to under-evaluation and incertitude in an important number of clinical cases. Our study was designed as a complex analysis of the stool specimens collected from the patients, in the purpose to improve the laboratory diagnostic and to enhance the number of confirmed cases of infectious diarrhoea. A number of 756 samples from inpatients with diarrhoea were tested targeting pathogenic and opportunistic bacteria, viruses and parasites by classical and molecular methods. We documented that, in case of non-Salmonella, non-Shigella, non-Yersinia diarrhoea, the quality of diagnostic was improved by increasing the percentage of positive specimens to 22.49% compared to 11.12% when only bacteria, 5.56% when only viruses and 4.10% when only parasites were investigated. The laboratory data are of great value in evaluating the diarrhoea syndrome offering the documentation for an accurate epidemiological response and an adequate treatment.

Correct complete denture rehabilitation, a chance for recovering abused tissues.

The prosthodontic treatment must provide for the edentulous patients bio-functional prosthetic restorations, bio-prophylactic for the surrounding tissues. In this aim, an edentulous patient must be submitted to a methodical clinical examination in order to establish the quality of hard and soft tissues, which will indicate the degree of difficulty of the prosthetic treatment. Additional investigation as a microbiologic examination and cephalometric radiographs can be useful in a modern investigation. In our daily practice, we are rarely confronted with a normal morphology of the denture bearing oral structures. The problem of managing abused tissues in a patient with morphologic abnormalities due to faulty prostheses is sometimes difficult to solve. Preventing the deterioration of oral status must be a condition in providing a chance for the success of the following rehabilitations, mainly in the situation when the complete edentulousness succeeds in a young or middle age patient.

Molecular detection of verotoxin-producing Escherichia coli isolates in stool samples from patients with diarrhea.

Despite its occurence as a commensal in the human intestine, Escherichia coli is also known as a versatile gastrointestinal pathogen. Identification of diarrheagenic E. coli (DEC) requires the accurate discrimination of pathogenic strains from commensal flora, and this is not an easy task if the diagnostic tools are inadequate. As the information regarding the relative contribution of DEC among other identifiable causes of infectious diarrhea in Romanian patients is scarce, a prospective study was conducted to evaluate the prevalence of enteropathogenic Escherichia coli (EPEC) and verotoxin-producing Escherichia coli (VTEC) isolates in the diarrheagenic stool specimens of 120 children and 270 adults. PCR-based detection of the eae, bfp, vtx1 and vtx2 genes was added to the conventional culture and slide agglutination with 12 commercial EPEC antisera and O157:H7 antisera for identifying EPEC and VTEC isolates. Even though E. coli colonies belonging to traditional EPEC serogroups were isolated from 35 children and 17 adults, only the isolates recovered from 16 children and 2 adults harboured at least one of the targeted pathogenicity-associated genes. The children shedding EPEC outnumbered the adults (16.7% vs. 7.4%). Based on the virulence genotype identified, the prevalence of atypical EPEC (eae+) was higher than of typical EPEC (eae+ bfpA+), and typical EPEC identification was restricted to children. Owing to the molecular analysis 5 children and 10 adults that could have been overlooked by the routine microbiological investigation were diagnosed as infected with VTEC. Considering that none of the screened stool specimens was positive for E. coli O157:H7, this study reports for the first time the presence of VTEC nonO157 in local patients with diarrhea. Our results bring evidence that both EPEC and VTEC isolates are circulating as agents of local sporadic cases of human diarrhea. Further studies are needed to evaluate the contribution of DEC to the human disease burden in Romania, based on improved diagnostic tools targeting the main virulence traits of E. coli clinical isolates.

Characteristics of Romanian fluoroquinolone-resistant human clinical Escherichia coli isolates.

Alarming progressive increase in the prevalence of antimicrobial resistance in Escherichia coli has been documented worldwide. Previous studies have suggested that many E. coli clinical isolates are actually low-virulence opportunists whose success derives more from antibiotic resistance than from pathogenic capability. The co-existence of ESBL production and fluoroquinolone resistance was reported as a major therapeutic challenge for E. coli infections. Considering the sparse information regarding the genetic background of virulence and antibiotic resistance of local isolates, a collection of ciprofloxacin-resistant E. coli isolates from human extraintestinal specimens was analyzed using PCR, PCR-sequencing, and PFGE, in order to clarify some aspects regarding their mechanisms of antimicrobial resistance, phylogenetic origin, the content of virulence-encoding determinants, and clonal relatedness. The tested fluoroquinolone resistant E. coli (FQREC) isolates, which displayed genetic heterogeneity, carried double mutations in the QRDR of gyrA previously described, which could explain their high resistance to ciprofloxacin. More than half of them (69%) possessed group 1 blaCTX. like genes, and with one exception, all these isolates were ESBL producers. The FQREC isolates belonging to non B2 phylogenetic groups outnumbered the isolates derived from B2 group (60 versus 27 isolates), and their overall content of virulence-encoding genes (fim, pap, sfa/foc, afa, hly, cnf and aer) was reduced. Regardless of the phylogenetic origin, the most prevalent virulence-associated genes possessed by the FQREC isolates were aer and fim determinants, while none of these isolates carried hly and cnf genes. In the case of weakened patients, the E. coli isolates do not need a robust virulence repertoire in order to overcome the host defense systems. The co-resistance of many FQREC isolates to extended-spectrum cephalosporins may provide a substantial advantage to their survival and spreading within the hospital environment.

Virulence characteristics of Escherichia coli isolates from children with urinary tract infections.

The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common infecting agent in children and adults of both sexes. In an attempt to evaluate the intrinsic virulence of E. coli uroisolates from children, 54 strains were assessed by using PCR for the presence of five representative genetic determinants coding for adherence systems (pap, sfa/foc, afa), and toxins (hly and cnf). The prevalence of pap, sfa/foc and afa genes was 55%, 54%, and 44%, respectively. Hemolysin-encoding gene hly was detected in 55% strains, while cnf was exhibited by 35% of the screened E. coli isolates. Among the 39 PCR positive strains isolated from children's urine cultures the co-occurrence of the various targeted virulence genes was detected in 30 strains, the virulence profiles identified suggesting the presence of their localization on chromosomal regions known as pathogencity-associated islands. The rapid and reliable detection of the intrinsic virulence potential by this molecular approach could be very useful when evaluating the importance of microorganism pathogenicity versus host's susceptibility for developing an overt symptomatology of infection.

A laboratory-based survey of Campylobacter infections in Prahova County.

In developing countries, as well as in many western countries, members of the genus Campylobacter are recognized as one of the most common cause of acute bacterial enteritis. Campylobacter jejuni and Campylobacter coli isolation rates have been shown to be equal to, and sometimes higher than those of other enteric pathogens. The Microbiology Laboratory of the local Public Health Authority in Prahova County conducted a one and a half-year laboratory-based survey of Campylobacter infections in patients suffering from gastrointestinal symptoms. From a total of 3284 stool samples screened, the culture-positive ones confirmed the bacterial etiology for 551 diarrhea cases. Campylobacter was found in 345 specimens, being the most frequently isolated enteropathogen. C. jejuni outnumbered C. coli species (239 vs. 106 isolates). Salmonella isolates were the second local cause of diarrhea. The highest isolation rate of Campylobacter was found in children 5 years of age (262 strains). The prevalence of campylobacteriosis declined with age. The isolation rate of Campylobacter (10.5%), the unimodal age-specific distribution of cases, as well as the identification of polymicrobial infections among the screened population were epidemiological aspects resembling reports on campylobacteriosis in developing countries. The susceptibility of Campylobacter isolates to various antimicrobial agents, including macrolides and fluoroquinolones was also assessed. Among the screened isolates, Erythromycin retained a good activity, while an increased ciprofloxacin resistance was observed. The information gathered through this local study sustains the importance of Campylobacter in the etiology of autochthonous infectious diarrhea. A development of a national surveillance program regarding the most important foodborne pathogens would be beneficial for improving prevention and controlling measures.

Genomic history of the seventh pandemic of cholera in Africa.

The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa.

Virulence genotypes of Escherichia coli strains from bacteremia in relation to phylogeny.

Bacteremia is the principal way of dissemination of local infections to distant organs. Escherichia coli bacteremia is almost always clinically significant, suggesting an increased risk of developing sepsis syndrome. Fifty-one E. coli bloodstream human isolates were analyzed using PCR technique for several molecular markers associated with extraintestinal virulence, and their phylogenetic group assignment, taking into account the link between the phylogenetic background and the intrinsic virulence of this species. Sixteen virulence genotypes have been identified, the majority of the blood isolates carrying the association of two genes. The genes encoding type 1 fimbria and aerobactin had the highest prevalence. As a confirmation of other studies, the strains assigned to E. coli phylogenetic group B2 exhibited the highest concentration of virulence genes, and represented almost half of the clinical blood isolates. The multifactorial virulence of E. coli strains isolated from invasive infections reflects a phylogenetic inheritance, and supports the concept of ExPEC pathotype as a subset of E. coli population involved in human infectious diseases. The surveillance of geographical variation of E. coli pathogenic clones is useful for epidemiological analysis.

Characterization of SPECTRUM Variable Nicotine Research Cigarettes.

OBJECTIVE: To provide researchers an extensive characterization of the SPECTRUM variable nicotine research cigarettes. METHODS: Data on cigarette physical properties, nicotine content, harmful and potentially harmful constituents in the tobacco filler was compiled. RESULTS: Data on physical properties, concentrations of menthol, nicotine and minor alkaloids, tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, ammonia, and toxic metals in the filler tobacco for all available varieties of Spectrum research cigarettes are provided. The similarity in the chemistry and physical properties of SPECTRUM cigarettes to commercial cigarettes renders them acceptable for use in behavioral studies. Baseline information on harmful and potentially harmful constituents in research tobacco products, particularly constituent levels such as minor alkaloids that fall outside typical ranges reported for commercial, provide researchers with the opportunity to monitor smoking behavior and to identify biomarkers that will inform efforts to understand the role of nicotine in creating and sustaining addiction. CONCLUSIONS: Well characterized research cigarettes suitable for human consumption are an important tool in clinical studies for investigating the physiological impacts of cigarettes delivering various levels of nicotine, the impact of reduced nicotine cigarettes on nicotine addiction, and the relationship between nicotine dose and smoking behavior.