investigating-effects-probiotics-on-pneumococcal-colonization-using

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an <em>In Vitro</em> Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Murdoch Childrens Research Institute

Multilocus sequence typing (MLST) is an important tool for the global surveillance of bacterial pathogens that is performed by comparing the sequences of designated housekeeping genes. We developed and tested a novel mass spectrometry-based method for MLST of Streptococcus pneumoniae. PCR amplicons were subjected to in vitro transcription and base-specific cleavage, followed by analysis of the resultant fragments by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Comparison of the cleavage fragment peak patterns to a reference sequence set permitted automated identification of alleles. Validation experiments using 29 isolates of S. pneumoniae revealed that the results of MALDI-TOF MS MLST matched those obtained by traditional sequence-based MLST for 99% of alleles and that the MALDI-TOF MS method accurately identified two single-nucleotide variations. The MADLI-TOF MS method was then used for MLST analysis of 43 S. pneumoniae isolates from Papua New Guinean children. The majority of the isolates present in this population were not clonal and contained seven new alleles and 30 previously unreported sequence types.

Multilocus sequence typing of Streptococcus pneumoniae by use of mass spectrometry.

The 7-valent pneumococcal conjugate vaccine (PCV7) reduces carriage of vaccine type Streptococcus pneumoniae but leads to replacement by nonvaccine serotypes and may affect carriage of other respiratory pathogens. We investigated nasopharyngeal carriage of S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian infants participating in a pneumococcal vaccine trial using quantitative PCR. Vaccination did not affect pathogen carriage rates or densities, whereas significant differences between the two major ethnic groups were observed.

Effect of pneumococcal vaccination on nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian children.

Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination.

Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

From 1985 to 1987, Streptococcus pneumoniae isolates were collected from children under 5 years of age in the Asaro Valley, Papua New Guinea as part of a study on bacterial colonization and respiratory tract infections. Data on serogroup and colony morphology were collected to survey serogroups and associated colony morphologies present in the area and to assess whether colony morphology can be indicative of serogroup. In total, 5989 colonies were examined; serogroups 6, 10, 14, 15, 19, 23, 33, 34, 35 and nonserotypeable strains were the most common and accounted for 77% of all the colonies, with serogroups 6, 19 and 23 accounting for 48%. The majority of colonies displayed the typical draughtsman morphology, though serogroup 10 and non-serotypeable isolates most often displayed a raised colony morphology. Of the 15 mucoid colonies identified 73% were serotype 3, though only 29% of serotype 3 isolates were mucoid. Thus colony morphology is of limited value in identifying the pneumococcal serogroup/serotype apart from mucoid colonies, which are likely to be serotype 3.

Streptococcus pneumoniae serogroups and colony morphology: a look back.

The current 'gold standard' for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping.

Production of latex agglutination reagents for pneumococcal serotyping.

The nasopharynx of children is often colonised by microorganisms such as Streptococcus pneumoniae (the pneumococcus) that can cause infections including pneumonia and otitis media. In this complex environment, bacteria and viruses may impact each other through antagonistic as well as synergistic interactions. Vaccination may alter colonisation dynamics, evidenced by the rise in non-vaccine serotypes following pneumococcal conjugate vaccination. Discovery of an inverse relationship between S. pneumoniae and Staphylococcus aureus carriage generated concern that pneumococcal vaccination could increase S. aureus carriage and disease. Here we review data on co-colonisation of pathogens in the nasopharynx, focusing on S. pneumoniae and the impact of pneumococcal vaccination. Thus far, pneumococcal vaccination has not had a sustained impact on S. aureus carriage but it is associated with an increase in non-typeable Haemophilus influenzae in acute otitis media aetiology. Advances in bacterial and viral detection methodologies have facilitated research in nasopharyngeal microbiology and will aid investigation of potential vaccine-induced changes, particularly when baseline studies can be conducted prior to pneumococcal vaccine introduction.

Nasopharyngeal microbial interactions in the era of pneumococcal conjugate vaccination.

Colonization of the nasopharynx by Streptococcus pneumoniae is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. Probiotic bacteria can influence disease outcomes through various mechanisms, including inhibition of pathogen colonization. Here, we examine the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on S. pneumoniae colonization of human epithelial cells using an in vitro model. We investigated the effects of LGG administered before, at the same time as, or after the addition of S. pneumoniae on the adherence of four pneumococcal isolates.

Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG.

Group A streptococcus (GAS) is the most common bacterial cause of sore throat. School-age children bear the highest burden of GAS pharyngitis. Accurate diagnosis is difficult: the majority of sore throats are viral in origin, culture-based identification of GAS requires 24-48 hours, and up to 15% of children are asymptomatic throat carriers of GAS. The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for detecting GAS pharyngitis and assess its suitability for clinical diagnosis.

Detection of group a streptococcal pharyngitis by quantitative PCR.

Bacterial isolates are often transported between laboratories for research and diagnostic purposes. Silica desiccant packets (SDPs), which are inexpensive and do not require freezing, were evaluated for storage and recovery of bacterial isolates. Conditions such as inoculum size, swab type and temperature of storage were investigated using ten Streptococcus pneumoniae isolates. The optimized protocol was then tested using 49 additional S. pneumoniae isolates representing 40 serogroups. Overall, S. pneumoniae growth was considered satisfactory (>100 colony forming units) for 98/109 (89.9%) and 20/20 (100%) swabs after 14 days at room temperature or 28 days at 4° C, respectively. Storage in SDPs did not impact on the ability of S. pneumoniae isolates to be subsequently serotyped. When the survival of nine other clinically relevant bacterial species was tested, seven were viable after 28 days at room temperature, the exceptions being Neisseria gonorrhoeae and Haemophilus influenzae. SDPs are suitable for transport and short-term storage of bacterial species including S. pneumoniae.

Article	Year
Multilocus sequence typing of Streptococcus pneumoniae by use of mass spectrometry. Journal of clinical microbiology\| PubMed ID: 21880964	2011
Effect of pneumococcal vaccination on nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian children. Journal of clinical microbiology\| PubMed ID: 22170924	2012
Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays. PloS one\| PubMed ID: 22470516	2012
Streptococcus pneumoniae serogroups and colony morphology: a look back. Papua and New Guinea medical journal\| PubMed ID: 23163187	2010
Production of latex agglutination reagents for pneumococcal serotyping. BMC research notes\| PubMed ID: 23379961	2013
Nasopharyngeal microbial interactions in the era of pneumococcal conjugate vaccination. Vaccine\| PubMed ID: 23523773	2013
Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG. BMC research notes\| PubMed ID: 23561014	2013
Detection of group a streptococcal pharyngitis by quantitative PCR. BMC infectious diseases\| PubMed ID: 23844865	2013
Silica desiccant packets for storage and transport of Streptococcus pneumoniae and other clinically relevant species. PloS one\| PubMed ID: 23940811	2013
Emergence of Streptococcus pneumoniae serotype 15A after the introduction of the conjugate vaccine in Victoria. The Medical journal of Australia\| PubMed ID: 24099202	2013

Eileen M. Dunne

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