Siamak Pour Yazdankhah

Distribution of Serogroups and Genotypes among Disease-Associated and Carried Isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway

ABSTRACT The distribution of serogroups and multilocus sequence types (STs) in collections of disease-associated and carried meningococci from the period 1991 to 2000 in three European countries (the Czech Republic, Greece, and Norway) was investigated. A total of 314 patient isolates and 353 isolates from asymptomatic carriers were characterized. The frequency distributions of serogroups and clone complexes differed among countries and between disease and carrier isolate collections. Highly significant differentiation was seen at each housekeeping locus. A marked positive association of serogroup C with disease was evidenced. The ST-11 complex was strongly positively associated with disease; associations for other clone complexes were weaker. The genetic diversity of the clone complexes differed. A single ST dominated the ST-11 clone complex, while the ST-41/44 complex exhibited greater levels of diversity. These data robustly demonstrated differences in the distribution of meningococcal genotypes in disease and carrier isolates and among countries. Further, they indicated that differences in genotype diversity and pathogenicity exist between meningococcal clone complexes.

Does the Wide Use of Quaternary Ammonium Compounds Enhance the Selection and Spread of Antimicrobial Resistance and Thus Threaten Our Health

Quaternary ammonium compounds (QACs) are widely used biocides that possess antimicrobial effect against a broad range of microorganisms. These compounds are used for numerous industrial purposes, water treatment, antifungal treatment in horticulture, as well as in pharmaceutical and everyday consumer products as preserving agents, foam boosters, and detergents. Resistance toward QACs is widespread among a diverse range of microorganisms and is facilitated by several mechanisms such as modifications in the membrane composition, expression of stress response and repair systems, or expression of efflux pump genes. Development of resistance in both pathogenic and nonpathogenic bacteria has been related to application in human medicine and the food industry. QACs in cosmetic products will inevitably come into intimate contact with the skin or mucosal linings in the mouth and thus are likely to add to the selection pressure toward more QAC-resistant microorganisms among the skin or mouth flora. There is increasing evidence of coresistance and cross-resistance between QACs and a range of other clinically important antibiotics and disinfectants. Use of QACs may have driven the fixation and spread of certain resistance cassette collectors (class 1 integrons), currently responsible for a major part of antimicrobial resistance in gram-negative bacteria. More indiscriminate use of QACs such as in cosmetic products may drive the selection of further new genetic elements that will aid in the persistence and spread of antimicrobial resistance and thus in limiting our treatment options for microbial infections.

Use of Variable-Number Tandem Repeats To Examine Genetic Diversity of Neisseria meningitidis

ABSTRACT Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of Neisseria meningitidis and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107 N. meningitidis isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s.

Zinc and copper in animal feed - development of resistance and co-resistance to antimicrobial agents in bacteria of animal origin.

Farmed animals such as pig and poultry receive additional Zn and Cu in their diets due to supplementing elements in compound feed as well as medical remedies. Enteral bacteria in farmed animals are shown to develop resistance to trace elements such as Zn and Cu. Resistance to Zn is often linked with resistance to methicillin in staphylococci, and Zn supplementation to animal feed may increase the proportion of multiresistant E. coli in the gut. Resistance to Cu in bacteria, in particular enterococci, is often associated with resistance to antimicrobial drugs like macrolides and glycopeptides (e.g. vancomycin). Such resistant bacteria may be transferred from the food-producing animals to humans (farmers, veterinarians, and consumers). Data on dose-response relation for Zn/Cu exposure and resistance are lacking; however, it seems more likely that a resistance-driven effect occurs at high trace element exposure than at more basal exposure levels. There is also lack of data which could demonstrate whether Zn/Cu-resistant bacteria may acquire antibiotic resistance genes/become antibiotics resistant, or if antibiotics-resistant bacteria are more capable to become Zn/Cu resistant than antibiotics-susceptible bacteria. Further research is needed to elucidate the link between Zn/Cu and antibiotic resistance in bacteria.

Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis

ABSTRACT A rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 × 106 CFU/ml for Staphylococcus aureus and 1 × 106 CFU/ml forEscherichia coli in spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.

Variable number tandem repeat typing of bacteria.

Analysis of bacterial genomes revealed a high percentage of DNA consisting of repeats, in which DNA motifs existed in multiple copies. Study of these DNA motifs has resulted in the development of variable number tandem repeat (VNTR) or multilocus variant-repeat analysis (MLVA) assays, which have shown to be valuable bacterial typing methods, especially in relation to disease outbreaks. The VNTR-based assay is based on direct PCR amplification of a specific locus, which is well defined. The range and polymorphism index of each locus can be calculated. This chapter describes the VNTR analysis of Neisseria meningitides-based on separation in low resolution media agarose, and VNTR analysis of Salmonella enterica subsp. enterica serovars Typhimurium-based on high resolution capillary electrophoresis.

Variable-Number Tandem Repeat Analysis of Meningococcal Isolates Belonging to the Sequence Type 162 Complex

ABSTRACT Thirty-one meningococcal isolates from carriers and disease cases belonging to the sequence type (ST) 162 complex, isolated in Greece in 1999 and 2000, were studied by the use of variable-number tandem repeat analysis. Our study demonstrated that the isolates belonging to the ST-162 clonal complex were a heterogeneous group. Based on this heterogeneity, it is unlikely that the disease-associated isolates represent an outbreak.

The use of probiotics for critically ill patients in hospitals

Abstract This article is based on a review of published literature, mainly from 2005 to 2008. Several reports demonstrate statistically significant beneficial health effects of probiotic supplementation, especially for the treatment of rotavirus-associated diarrhoea. The following main topics have been reviewed: bacterial translocation from the gut and infectious disease; virulence factors including toxicity; metabolic functions including host storage, platelet aggregation, deconjugation of bile acids, and degradation of mucin. Furthermore, resistance to antimicrobials, with emphasis on the location of the genes involved, has been reviewed. Studies concerning probiotic supplementation in hospital patients suffering from acute pancreatitis, antibiotic-associated diarrhoea, including Clostridium difficile infection, and non-alcoholic fatty liver disease are reviewed. Supplementation with probiotic bacteria in critically ill children, e.g. in intensive care, is also discussed. The use of probiotics for patients with diarrhoea, Helicobacter pylori infection and inflammatory bowel disease is mentioned, as well as for patients with AIDS, urogenital infections, and small intestinal bacterial overgrowth. Particular attention has been paid to a large Dutch study (PROPATRIA), which showed increased mortality in patients with acute pancreatitis who were given enteral nutrition to which probiotics had been added, compared with enteral nutrition alone. Similarities in some acquired resistance genes between probiotic microorganisms and other bacteria of human origin suggest the transfer of resistance genes between commensal microorganisms in the complex gastrointestinal tract ecosystem. Intake of probiotic microorganisms that carry transferable antimicrobial resistance genes may increase the risk of the transfer of such genes to the resident microbiota in patients. Although some beneficial effects have been reported in some patient groups, we conclude that the adverse effects that have been observed are well documented, thus indicating that caution should be observed in administering probiotic bacteria to critically ill patients.

The history of antibiotics

The development of chemical compounds for the treatment of infectious diseases may be divided into three phases: a) the discovery in the 1600s in South America of alkaloid extracts from the bark of the cinchona tree and from the dried root of the ipecacuanha bush, which proved effective against, respectively, malaria (quinine) and amoebic dysentery (emetine); b) the development of synthetic drugs, which mostly took place in Germany, starting with Paul Ehrlichs (1854-1915) discovery of salvarsan (1909), and crowned with Gerhard Domagks (1895-1964) discovery of the sulfonamides (1930s); and c) the discovery of antibiotics. The prime example of the latter is the development of penicillin in the late 1920s following a discovery by a solitary research scientist who never worked in a team and never as part of a research programme. It took another ten years or so before drug-quality penicillin was produced, with research now dependent on being conducted in large collaborative teams, frequently between universities and wealthy industrial companies. The search for new antibiotics began in earnest in the latter half of the 1940s and was mostly based on soil microorganisms. Many new antibiotics were discovered in this period, which may be termed «the golden age of antibiotics». Over the past three decades, the development of new antibiotics has largely stalled, while antibiotic resistance has increased. This situation may require new strategies for the treatment of infectious diseases.

Assessment of Probiotics in Infant Formula and Cereal Based Baby Foods Containing Bifidobacterium lactis Bb12– Update 2014

“Assessment of benefits and risks of probiotics in processed cereal-based baby foods supplemented Bifidobacteriumn lactis Bb12” from 2010 answered a request from the Norwegian Food Safety Authority focusing on the age groups 4-6 months, 6-12 months and 1-3 years. However, the use of infant formula intended for newborns, supplemented with this probiotic, was neither asked by the NFSA nor assessed by VKM. The notifier of the baby foods intended for infants and small children has provided information on three different cereal-based products intended for age-groups over 4 months and one infant formula intended for newborns, all supplemented with B. lactis. In its letter the company concludes that their products supplemented with B. lactis do not pose any health and safety risk. Regarding health effect, we have already mentioned in our assessment (Halvorsen et al. 2010) that: “It is not the mandate of this report to evaluate the health claims related to the products as these health claims are assessed by EFSA.” Our main conclusions regarding safety were as follows: “No serious adverse events are reported, but neither has the effect of long-term intake of a single Grey Literature Lassen and Yazdankhah; EJNFS, 5(2): 101-103, 2015; Article no.EJNFS.2015.008 102 bacterial strain been studied. Furthermore, cereals supplemented with B. lactis Bb12 intended for infants and toddlers have not been studied regarding safety. We are not aware of any in vivo studies explicitly concerning the ability of B. lactis Bb12 to influence gene expression of epithelial cells”. Furthermore, we were concerned regarding presence of antibiotic resistance gene against tetracycline (tetW) in the B. lactis Bb12. In the answer to the question from NFSA regarding antibiotic resistance gene in L. lactis Bb12, we concluded that: “Consumption of probiotic microororganism B. lactis Bb12 that harbour gene encoding resistance against tetracycline (tetW) may increase the risk of the transfer of such genes to the resident microbiota and pathogenic bacteria and hence increase development of bacterial resistance. High similarity has been observed between tetW gene in bacteria of human and environment origin and B. lactis Bb12. This suggests the spread of tetracycline resistance gene (tetW) between bacteria of various origins. However, the transfer of tetracycline resistance gene (tetW) to other bacteria as a consequence of consumption of Bb12 has not been studied.” As we have already mentioned in our assessment (Halvorsen et al. 2010). “It is important to note that the infant’s diet comprises a restricted variety of foods, which often are taken several times a day during a period of life when a stable intestinal flora is not yet established. The establishment of a normal intestinal microbiota takes at least two years and thus the intake of large numbers of probiotic bacteria in monoculture during the first years of life may greatly influence this process.” According to the “Guideline for evaluation of probiotics in food” (FAO/WHO 2002): ‘‘....the onus is on the producer to prove that any given probiotic strain is not a significant risk with regard to transferable antibiotic resistance or other opportunistic virulence properties.” The tet(W) gene in Bifidobacterium seems to be integrated in the chromosome and its surrounding regions vary depending on the strain, but very often the gene is flanked by transposase target sequences or genes coding for transposase, an enzyme that catalyzes the movement of DNA fragments between different locations by recognizing specific target sequences, suggesting that, under adequate conditions, the gene could be transferred (Gueimonde et al. 2013). The presence of a tetracycline resistance gene, tet(W), flanked by a putative transposase gene in B. animalis subsp. lactis was also confirmed in other strains of Bifidobacterium than Bb12 (Stahl & Barrangou 2012). Among the data provided by the notifier, we could not identify any new studies regarding the above mentioned concerns. As already mentioned, our assessment from 2010 did not include probiotic-supplemented infant formula intended for use by newborns. It seems likely that the same concerns as for the cerealbased products will be valid in this age group and possibly of even greater importance. Among the literature provided by the notifier was also the position paper from 2011 of the ESPGHAN Committee on Nutrition (ESPGHAN 2011). Among their general conclusions are: • (Conclusion 1): “For healthy infants, the available scientific data suggest that the administration of currently evaluated probiotic-supplemented formula to healthy infants does not raise safety concerns with regard to growth and adverse effects”. But none the less: • (Conclusion 5): “In general, there is a lack of data on the long-term effects of the administration of formula supplemented with probiotics. Such data would be of particular importance if the effects persisted after the administration of the probiotics has ceased.” And concludes lastly; Lassen and Yazdankhah; EJNFS, 5(2): 101-103, 2015; Article no.EJNFS.2015.008 103 • (Conclusion 6): “Considering the above, the Committee does not recommend the routine use of probiotic-supplemented formula in infants.” Our view is in accordance with these conclusions.