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Introduction: Streptococcus pneumoniae can be responsible for severe human infections. Optochin resistance has been a potential cause of misidentification of pneumococcus and other members of the mitis group. Hence, rapid and easy optochin resistant (Optr) S. pneumoniae identification is essential. Methodology: Atypical pneumococci were characterized using optochin susceptibility, bile solubility based on spectrophotometric reading, serotyping, pulsed field gel electrophoresis (PFGE), 16S rRNA sequencing and PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802 and Spn9828. Results: Optical density values for the bile solubility test suggest the identification of four Optr S. pneumoniae and one Streptococcus pseudopneumoniae. All Optr pneumococci harbored cpsA, lytA, ply, Spn9802, Spn9828 and pspA genes. Only ply, spn9802 and Spn9828 genes were detected in S. pseudopneumoniae. The 16S rRNA sequencing differentiates between these two species. Optr S. pneumoniae strains belonged to different genotypes and serotypes (14, 19A, 3 and 9V). Three Optr S. pneumoniae isolates were typed as pspA family 2, while one belonged to pspA family 1. Sequencing of the atpA and atpC gene of the Optr variants revealed three mutations in the ATPase a-subunit (L99I, M23V and V52I) and one mutation in ATPase c-subunit (V48I). Conclusions: Our data indicate that bile OD-values provides an accurate, fast and easy method to discriminate between Optr S. pneumoniae and other Streptococcus mitis group. Moreover molecular techniques, confirming the bile test, can be used in order to prevent these atypical pneumococci and alert clinical microbiologists of the presence of these strains in the community.

Streptococcus pneumoniae are gram positive normal flora of the upper respiratory tract in humans but are also the primary causative bacterial agents of pneumonia and otitis media mostly in children. They are also known to cause sinusitis, bronchitis, meningitis as well as bacteremia. They are differentiated from other Streptococci by their special characteristics such as their morphology, bile solubility as well as their sensitivity to optochin in addition to their display of a capsule. They are sensitive to most antibiotics and the beta lactams are considered the best drugs of choice in their treatment. Antimicrobial resistance currently considered as a serious public health challenge, not only in hospital settings but also in the community at large. Aims and Objectives 1. To find out the occurrence of pneumococcal etiology in Community Acquired Pneumonia (CAP) and Hospital Acquired Pneumonia (HAP). 2. To find out Penicillin resistance among these isolates by: Penicillin E-strips. 3. To identify Macrolide resistance among these isolates by: Erythromycin E- strips. Materials and Methods: Pneumococcal isolates were collected from the respiratory samples such as sputum, endotracheal secretions, throat swab, broncho alveolar lavange, and also from other samples such as ear swab and the bacteremia suspected blood samples. Results: A total of 30 pneumococcal isolates were collected from respiratory samples namely: sputum, endotracheal secretions, throat swab, broncho alveolar lavange, and other samples like ear swab and blood. The collected isolates were confirmed to be Streptococcus pneumonia. Out of 30 samples, 18 (60%) were collected from male patients and 12 (40%) samples from female patients. Conclusion: The need of MIC determination for antimicrobial susceptibility pattern testing of Pneumococci is a better method because it was found that the isolates which were sensitive for disc diffusion method even had the resistant MIC as 40% and 56.6% for Penicillin and Erythromycin. Keywords: Streptococcus pneumonia, Optochin sensitive, Antimicrobial resistance.

BACKGROUND: Streptococcus pneumoniae remains a major contributor to childhood infections and deaths globally. In Cameroon, the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in July 2011, using a 3-dose Expanded programme on immunization (EPI) schedule administered to infants at 6, 10 and 14 weeks of age. To evaluate PCV13 effects, we assessed pneumococcal nasopharyngeal colonization and serotype distribution among Cameroonian children after PCV13 introduction. METHODS: Nasopharyngeal (NP) swabs were collected from eligible children aged 24-36 months in two cross-sectional surveys conducted from March to July: in 2013 (PCV13-unvaccinated), and in 2015 (PCV13-vaccinated). Using a systematic World Health Organization (WHO) cluster coverage sampling technique in 40 communities, NP swabs collected were processed following WHO recommendations. Standard bacterial culture techniques were used for the isolation of S. pneumoniae from gentamicin-blood agar plates and identification using optochin susceptibility testing. Serotyping was performed using sequential multiplex polymerase chain reaction, supplemented with Quellung test. RESULTS: Among the PCV13-vaccinated children, overall pneumococcal carriage prevalence was 61.8% (426/689) and PCV13 vaccine-type carriage prevalence was 18.0% (123/689). Eleven out of the 13 vaccine serotypes were detected in the vaccinated children. The most common serotypes were 19F (4.5%, 31/689) and 15B/C (7.3%, 50/689). CONCLUSION: In Cameroon, four years after infant vaccination nearly all of the PCV13-serotypes continued to circulate in the population. This suggests that the direct and indirect effects of the vaccination programme have not resulted in expected low levels of vaccine-type transmission. Continuous monitoring is needed to assess the long term effects of the PCV13 on nasopharyngeal carriage and disease. publishedVersion

Streptococcus pneumoniae is a highly adept human pathogen. A frequent asymptomatic member of the respiratory microbiota, the pneumococcus has a remarkable capacity to cause mucosal (pneumonia and otitis media) and invasive diseases (bacteremia, meningitis). In addition, the organism utilizes a vast battery of virulence factors for tissue and immune evasion. Though recognized as a significant cause of pneumonia for over a century, efforts to develop more effective vaccines remain ongoing. The pathogen’s inherent capacity to exchange genetic material is critical to the pneumococcus’ success. This feature historically facilitated essential discoveries in genetics and is vital for disseminating antibiotic resistance and vaccine evasion.

Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL, correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study, however, confirmation and further improvements are warranted.

Aim. To investigate optochin-resistant viridans group streptococci (VGS) strains isolated from the sputum sample of adult patients with different clinical symptoms. Materials and Methods. Optochin-resistant VGS isolates were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). recA sequencing was used to confirm identified isolates at the genus level by MALDI-TOF MS. Finding. We identified 79% of tested isolates (148/187) at the species-level identification using the MALDI-TOF MS tool. We identified that the most common species isolated from sputum specimens were S. oralis (44.9%) followed by S. mitis (25.7%), S. infantis (9.1%), S. parasanguinis (7.5%), S. peroris (3.7%), S. anginosus (2.7%), and S. sanguinis (2.1%). Discussion. The S. oralis strains were majority of optochin-resistant VGS isolates obtained from sputum of adult patients in Jakarta, Indonesia. MALDI-TOF MS showed potential for the rapid identification tool to identify optochin-resistant VGS isolates. Although there were discrepancies in identifying isolates at the genus/species level, the performance could be improved by expanding its database.

Background Streptococcus pneumoniae S pneumoniae is the main cause of febrile conditions especially among children below two years and the elderly above years old worldwide In Cameroon the frequency of S pneumoniae as the cause of meningitis varied from to from to The aim of this study was to determine the susceptibility to antibiotics of S pneumoniae involved in meningitis nbsp Methods A cross sectional study was carried out during a four year period from April to March Cerebrospinal fluid CSF was collected by clinicians from children less than years with clinically suspected bacterial meningitis at the Mother and Child Center Chantal Biya Foundation MCC CBF in Yaounde A probable case is a suspected clinical case with the CSF showing at least one of the following elements turbid appearance leukocytes ge mm elevated protein gt mg dl or decreased glucose lt mg dl Cases are confirmed when isolates are identified The identification of S pneumoniae was done by soluble antigens the Binax Now reg test Gram stain catalase test optochin test bile solubility and molecular serotyping The antimicrobial susceptibility testing was carried out using the disc diffusion method on Mueller Hinton medium with sheep blood and the minimal inhibitory concentration was found using the Etest reg nbsp Results Out of clinically suspected cases of bacterial meningitis were probable and cases were confirmed Most were isolated in children less than one year old and in boys These incidences occurred mainly during the dry season In the confirmed cases were due to S pneumoniae due to Haemophilus influenzae B due to Neisseria meningitis and due to other germs Among the serotyped pneumococci belonged to serotype A B Of viable isolates of S pneumoniae were resistant to penicillin G and to ceftriaxone nbsp Conclusion S pneumoniae is the main etiology of childhood meningitis in Yaounde The frequency of resistance to penicillin G was high while moderate to ceftriaxone

Background The study objective was to determine the carriage and serotype distribution of Streptococcus pneumoniae among children in Accra, Ghana, five years after the introduction of the pneumococcal conjugate vaccine (PCV-13) in 2012. Methods Nasopharyngeal swab samples were collected from 410 children below 5 years of age in Accra, Ghana, from September to December, 2016. Pneumococcal isolates were identified by optochin sensitivity and bile solubility. Serotyping was performed using the latex agglutination kit and Quellung reaction. The isolates were furthermore tested for antimicrobial susceptibility for different antimicrobials, including penicillin (PEN). Twelve isolates including seven non-typeable (NT) isolates were characterized using whole-genome sequencing analysis (WGS). Results The overall carriage prevalence was found to be 54% (95% CI, 49–59%), and 20% (95% CI, 49–59%) of the children were carrying PCV-13 included serotypes, while 37% (95% CI, 33–42%) of the children were carrying non-PCV-13 serotypes. Based on the serotype distribution, 33% of all observed serotypes were included in PCV-13 while 66% were non-PCV-13 serotypes. The dominating non-PCV-13 serotypes were 23B, 16F, and 11A followed by PCV-13 serotypes 23F and 19F. The PCV-13 covers the majority of resistant isolates in Accra. A proportion of 22.3% of the isolates showed intermediate resistance to penicillin G, while only one isolate showed full resistance. Forty-five isolates (20.5%) were defined as multidrug-resistant (MDR) as they were intermediate/resistant to three or more classes of antimicrobials. Of the seven NT isolates characterized by WGS, four showed highest match to genotype 38, while the remaining three showed highest match to genotype 14. Four MDR serotype 19A isolates were found to be MLST 320. Conclusion PCV-13 introduced in Ghana did not eliminate PCV-13 covered serotypes, and the carriage rate of 54% in this study is similar to carriage studies from pre PCV-13 period. However, the penicillin non-susceptible isolates have been reduced from 45% of carriage isolates before PCV-13 introduction to 22.3% of the isolates in this study. Continuous monitoring of serotype distribution is important, and in addition, an evaluation of an alternative vaccination schedule from 3 + 0 to 2 + 1 will be important to consider. Electronic supplementary material The online version of this article (10.1186/s12887-019-1690-5) contains supplementary material, which is available to authorized users.

Background Streptococcus pneumoniae identification has traditionally been based on two biochemical tests, susceptibility of pneumococci to optochin and solubility in bile-salt solution. Due to slowness and sometimes difficulty in interpretation, the bile solubility test has fallen into disuse. The main objective of this work was to assess the current effectiveness of the optochin susceptibility test in pneumococcal identification in clinical practice. Materials and methods Overall 126 viridans group streptococci consecutively isolated from respiratory samples were analyzed using the optochin susceptibility test by picking one colony from the culture. Sixty-two were initially considered optochin susceptible, and 64 were considered optochin resistant and analyzed with the bile solubility test. If a discrepancy between the tests was observed (i.e., whether an isolate was optochin susceptible and bile insoluble or optochin resistant and bile soluble), then the optochin susceptibility test was repeated, adjusting the inoculum to a McFarland standard of 0.5. Species were identified by sequencing the lytA and recA genes. Results Twelve discrepancies were initially observed. The result of the repeated optochin test showed that the initial optochin test of 4 isolates had been wrongly interpreted. Of the remaining 8 discrepancies, 2 optochin-resistant bile-soluble isolates were identified by gene sequencing as S. pneumoniae, and of the 6 optochin-susceptible bile-nonsoluble isolates, 3 were identified as Streptococcus mitis and 3 as Streptococcus pseudopneumoniae. Conclusions The optochin test correctly identified 90.5% of all recent viridans group streptococci clinical isolates which include both optochin susceptible (62/126 = 49.2%) and optochin resistant (64/126 = 50.8%) strains. Of the group of optochin susceptible viridans, 87.5% were correctly identified, and 93.5% of the optochin resistant group were correctly identified. However, this technique does not correctly differentiate between S. pneumoniae from other viridans group streptococci in the clinical setting. Additional testing is needed for that identification.

Purpose: In Turkey, pneumococcal conjugate vaccine (PCV) was introduced to the national immunization program as PCV7 in 2008, and was replaced with PCV13 in 2011. The aim of the study was to demonstrate the pneumococcal carriage rate and the serotype distribution in healthy children under 5 years in Turkey who were vaccinated with PCV13. Methods: We conducted a cross-sectional study including the collection of questionnaire data and nasopharyngeal (NP) specimens among children aged, Pfizer [WI229479], The study was supported by Pfizer [grant number WI229479].

Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Optr) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the F0F1ATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Optr isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Optr isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Optr and Opts subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Optr strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.

Background Streptococcus pneumoniae is a bacterial pathogen that colonizes the human nasopharynx. Colonization is frequently reported to be high in young children. In this study, we investigated the nasopharyngeal (NP) carriage rate, serotype distribution, and antibiotic susceptibility of S. pneumoniae in children under five years of age in Kotabaru, South Kalimantan, Indonesia. Methods NP swab specimens were collected from 399 young children (mean age: 30 months) who participated in the Rampa Village Community Health Center, with 74% of the participants being Bajau children. S. pneumoniae was identified using optochin susceptibility and bile solubility tests. Serotyping was performed by sequential multiplex PCR, and antimicrobial susceptibility profiling was performed by disk diffusion and microdilution methods. Results The NP carriage rate of S. pneumoniae was 45% (180/399). The most commonly serotypes were 6A/6B (18%), followed by 15B/15C (17%), 19F (16%), 34 (8%), and 23F (5%); 46% of them were identified as strains of the PCV13 vaccine type. Additionally, almost half of the pneumococcal isolates were non-susceptible to penicillin (40%), whereas non-susceptibility to tetracycline (36.8%), trimethoprim/sulfamethoxazole (29.7%), erythromycin (16.8%), chloramphenicol (9.7%), and clindamycin (8.6%) was also found. We identified 18% (n = 34) of S. pneumoniae isolates as multidrug-resistant (MDR) strains, and serotype 19F was the most common (74%) among them. Conclusions MDR S. pneumoniae vaccine type strains were dominated by serotype 19F. The implementation of a pneumococcal conjugate vaccine program in Indonesia might reduce MDR strains circulating in the community in the future.

The Mitis group of streptococci includes an important human pathogen, Streptococcus pneumoniae (pneumococcus) and about 20 other related species with much lower pathogenicity. In clinical practice, some representatives of these species, especially Streptococcus pseudopneumoniae and Streptococcus mitis, are sometimes mistaken for S. pneumoniae based on the results of classical microbiological methods, such as optochin susceptibility and bile solubility. Several various molecular approaches that address the issue of correct identification of pneumococci and other Mitis streptococci have been proposed and are discussed in this review, including PCR- and gene sequencing-based tests as well as new developments in the genomic field that represents an important advance in our understanding of relationships within the Mitis group.

The aim of our study was to investigate phenotypic and genotypic features of streptococci misidentified (misID) asStreptococcus pneumoniae, obtained over 20 years from hospital patients in Poland. Sixty-three isolates demonstrating microbiological features typical for pneumococci (optochin susceptibility and/or bile solubility) were investigated by phenotypic tests,lytAand 16S rRNA gene polymorphism and whole-genome sequencing (WGS). All isolates had a 6-bp deletion in thelytA3′ terminus, characteristic for Mitis streptococc and all but two isolates lacked the pneumococcal signature cytosine at nucleotide position 203 in the 16S rRNA genes. The counterparts ofpsaAandplywere present in 100% and 81.0% of isolates, respectively; the spn9802 and spn9828 loci were characteristic for 49.2% and 38.1% of isolates, respectively. Phylogenetic trees and networks, based on the multilocus sequence analysis (MLSA) scheme, ribosomal multilocus sequence typing (rMLST) scheme and core-genome analysis, clearly separated investigated isolates fromS. pneumoniaeand demonstrated the polyclonal character of misID streptococci, associated with theStreptococcus pseudopneumoniaeandStreptococcus mitisgroups. While theS. pseudopneumoniaeclade was relatively well defined in all three analyses, only the core-genome analysis revealed the presence of another cluster comprising a fraction of misID streptococci and a strain proposed elsewhere as a representative of a novel species in the Mitis group. Our findings point to complex phylogenetic and taxonomic relationships amongS. mitis-like bacteria and support the notion that this group may in fact consist of several distinct species.

Purpose: The aims of this study were to characterize alpha-hemolytic streptococci among isolates from cases of infectious endophthalmitis and keratitis and to determine their distributions. Methods: The sample included 27 and 35 nonduplicated isolates of alpha-hemolytic streptococci recovered from patients with infectious endophthalmitis (2002-2013) and keratitis (2008-2013), respectively. Isolates were identified by the optochin susceptibility and bile solubility tests, using a biochemical identification system. The minimum inhibitory concentration was determined by the broth microdilution method. Molecular identification was performed by analyses of three constitutive genes and the complementary multilocus sequence. The molecular epidemiology of Streptococcus pneumoniae was investigated using multilocus sequence typing, and the presence of the capsular polysaccharide-encoding gene was assessed using conventional polymerase chain reaction. Outcomes were evaluated using the patients’ medical records. Results: Phenotypic tests differentiated S. pneumoniae from other alpha-hemolytic streptococci, consistent with later molecular identifications. Streptococcus oralis was significantly prevalent among the endophthalmitis isolates, as was S. pneumoniae in the keratitis isolates. High levels of susceptibility to antibiotics were observed, including vancomycin, cephalosporins, and fluoroquinolones. High genetic variability was detected among the 19 S. pneumoniae strains, with 15 predicted to be encapsulated. The medical records of patients with infectious endophthalmitis were reviewed (n=15/27; 56%), and final visual acuity was assessed in 12 cases (44%). Many patients progressed to a final visual acuity state of “no light perception” (6/12; 50%), “light perception” (3/12; 25%), or “hand motion” (1/12; 8%). The medical records of patients with infectious keratitis were also reviewed (n=24/35; 69%), and final visual acuity was assessed in 18 cases (51%). Similarly, most patients progressed to a final visual acuity state of “no light perception” (6/18; 33%), “light perception” (1/18; 6%), or “hand motion” (6/18; 33%). Overall, the majority of patients progressed to a final visual acuity state of “no light perception” (12/30), “light perception” (4/30), or “hand motion” (7/30). Conclusions: The distribution of alpha-hemolytic streptococci in ocular infections suggested the presence of a species-specific tissue tropism. The prognoses of patients with ocular streptococcal infections were highly unfavorable, and antibiotic resistance did not contribute to the unfavorable clinical progressions and poor outcomes.

Streptococcus pseudopneumoniae is a mischaracterized species of Streptococci that is usually overlooked during examination of sputum samples of patients with chest infections. The association of this organism with lower respiratory tract diseases is still unclear and its isolation and description is underestimated in our locality. To our knowledge, there are no published studies on the isolation of S. pseudopneumoniae from pathological specimens in Egypt. The aims of this study were to isolate S. pseudopneumoniae from sputum specimens of patients admitted to Chest Department of Mansoura University Hospitals (MUHs) and to differentiate it from Streptococcus pneumoniae and other viridans group Streptococci, also to determine its prevalence and associated risk factors. Sixteen isolates of S. pseudopneumoniae were diagnosed phenotypically by optochin susceptibility and bile solubility tests followed by genotypic characterization by multiplex polymerase chain reaction (PCR). All of the isolates were subjected to antibiotic susceptibility testing using the disk diffusion method. The prevalence of S. pseudopneumoniae among studied patients was 4.9% (16/329). All of the examined isolates were found to be positive for aliB-like ORF2 and negative for cpsA and lytA genes by multiplex PCR. Elevated resistance rate of the isolates was recorded for erythromycin, penicillin and co-trimoxazole. Infection by S. pseudopneumoniae was found to be significantly associated with Chronic Obstructive Pulmonary Diseases (COPD). Although, S. pseudopneumoniae recorded a low prevalence in this study, their elevated antibiotic resistance together with the association with COPD and their pure isolation from sputum samples underline the necessity of spending more effort for their detection and characterization in the microbiology laboratories. Key words: Chronic obstructive pulmonary diseases (COPD), optochin, respiratory diseases, Streptococcus pseudopneumoniae.

Purpose. Optochin susceptibility is one parameter used in the laboratory to identifyStreptococcus pneumoniae. However, a single standardized procedure does not exist. Optochin is included neither in the current EUCAST breakpoint tables nor in the CLSI performance standards for antimicrobial susceptibility testing. We wanted to establish an evidence-based protocol for optochin testing for our Total Lab Automation.Methods. We tested seven different agars and four different reading time points (7 h, 12 h, 18 h, and 24 h). To accommodate for serotype diversity, all tests were done with 99 different strains covering 34 different serotypes ofS. pneumoniae. We calculated a multivariable linear regression using data from 5544 inhibition zones.Results. Reading was possible for all strains at 12 h. Agar type and manufacturer influenced the size of the inhibition zones by up to 2 mm and they varied considerably depending on serotype (up to 3 mm for serotype 3). Depending on agar and reading time point, up to 38% of inhibition zones were smaller than the cut-off of 14 mm; that is, the result of the test was false-negative.Conclusions. Shortening incubation time from 24 h to 12 h for optochin susceptibility testing is feasible. Agar and incubation time have to be chosen carefully to avoid false-negative results.

Pneumonia with bacteremia accounts for the majority (70%) of cases of invasive pneumococcal diseases. Laboratory characterization of pneumococci is routinely done by sensitivity to optochin. We identified an atypical presentation of optochin-resistant pneumococci in blood isolate of a patient with sepsis. Polymerase chain reaction for autolysin (lytA) and pneumolysin (ply) was positive in the bile soluble isolate. Gene sequencing revealed synonymous single nucleotide polymorphism type of functional mutation in lytA gene. There have been emerging reports of optochin-resistant pneumococci. Diminishing relevance and reliability of optochin sensitivity testing in identifying pneumococci in clinical samples has been highlighted by this case report.

A general survey for six months was undertaken for the prevalence of environmental bacterium Streptococcus pneumoniae among the different age groups (3-65 years) including both sexes from various hospitals of Hyderabad city. Laboratory examinations revealed S. pneumoniae as most potent environmental pathogen from the sputum and throat swabs of old aged patients and children respectively. During observations, 39 specimens were growth positive; the biochemistry of isolates revealed that they were coagulase, catalase and oxidase negative, TSI, gel hydrolysis positive and were able to ferment glucose, lactose, maltose, galactose, fructose, sucrose, starch and raffinose. The results of antimicrobial activity showed that pneumococci were resistant to the cefspan, septran, cravit, pipemetic acid, azomax, bacitracin, and penicillin and a clear zone of inhibition was observed on clithromycin, optochin, cefizox, genatamycin, minocyclin, levoflaxacin, and vancomycin. There were intermediate zone of inhibition found on claforan, nalidixic acid, amoxycillin, fosfomycin, fortum, and erythromycin on Mueller Hinton’s agar after 24 hours incubation.

Streptococcus pneumoniae is a rare cause of urinary tract infection. Between January 2010 and December 2014, 26 urine samples from 18 different patients contained S. pneumoniae at the Department for Infectious Diseases, University Hospital of Heidelberg. Patient age varied between three and 72 years. 13 patients were male and five were female. Past medical histories of 16 patients were available. Eight patients had a past medical history of renal transplant and four patients had other renal dysfunctions. Further analyses of the isolates revealed that the aspect of colonies is more resembling S. mitis than invasive isolates of S. pneumoniae. Optochin disk diameters tend to be 14 mm or smaller. Identification using MALDI-TOF or VITEK2 identification cards was accurate. Only 2 isolates showed a decreased susceptibility towards penicillin (MIC = 0.5 mg/L). Eight different serotypes were identified using a PCR approach as well Neufeld-Quellungs reaction.

Streptococcus pseudopneumoniae was described in 2004 as a new human pathogen, acknowledged in a range of clinical infections typically associated to the respiratory tract. This report demonstrates that S. pseudopneumoniae has the potential to cause invasive infection. In blood cultures from three patients, growth of an atypical Streptococcus pneumoniae (non-capsular, non-serotypeable, optochin susceptible under ambient atmosphere and bile-intermediately soluble) was recovered. All three patients had a history of a haematological disease (myelodysplastic syndrome and multiple myeloma) and an apparent origin of infection related to the liver or bile duct. All isolates were genome sequenced and subsequently identified as S. pseudopneumoniae by multi-locus sequence analysis (MLSA). Multi-locus sequence typing (MLST) based on the S. pneumoniae scheme revealed unknown sequence types and the antibiogram and resistome revealed no antibiotic resistance.

Aim: The objective of this study is to compare various Streptococcus pneumoniae identification methods. Materials & methods: In total, 1371 putative S. pneumoniae isolates were tested with three phenotypic methods and a molecular-based method targeting a virulence factor (CpsA). We assessed the sensitivity and the specificity of each method and widely used S. pneumoniae identification algorithm. Results: None of the methods or the identification algorithm used separately was able to correctly identify all S. pneumoniae isolates. Furthermore, a high rate of optochin resistance was found. Conclusions: We demonstrated the failure of the current S. pneumoniae identification methods and optochin susceptibility-based algorithm. In addition, the high rate of optochin resistance might justify the necessity of a close monitoring of optochin susceptibility.

Introduction: Streptococcus pneumoniae remains as a major causative agent for morbidity and mortality cases in the world. The pathogenicity of this bacterium is attributed to many virulence factors. These factors, though not all had been extensively investigated at the molecular level in various mutant studies. However, of these studies, none of them were using local Malaysian isolate. Materials and Methods: Therefore, to better understand the growth pattern of local mutants a growth curve study and optochin disk susceptibility test were conducted. Several virulence genes (i.e hyl, ugl, ccpA, nanA, and ply) were targeted for this local mutant construction. Pneumococcal mutants were constructed by employing genome-wide deletion technique using high throughput polymerase chain reaction (PCR). Linear knockout construct was established by fusing the kanamycin resistance protein encoding gene, aphA-3 with 1 Kb homologous sequence up and down stream of the respective targeted genes. The gene was knocked out by allelic replacement mechanism. Results: Glycosaminoglycan (GAG) gene cluster mutants (∆hyl- and ∆ugl-) took two days compared to 3 others to grow on brain heart infusion (BHI)-kanamycin agar plate following transformation. This indicated a variance in the mutant growth pattern. Therefore, a growth curve study was conducted to validate the previous observation. We report different time span of log phase in growth curve pattern of mutants relative to the wild type strain. Meanwhile, comparison of zone of inhibition using optochin disk showed non-identical inhibition diameter (≥ 14mm) corresponding to the wild type (14.3mm). Conclusion: The difference in growth pattern was attributed to nutritional role of certain gene.

This study examined the carriage of Streptococcus pneumoniae in healthy Danish children aged 8–19 months and assessed the effect of the probiotics Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp lactis on the pneumococcal carriage during daycare enrolment. Potential risk factors of pneumococcal carriage were analysed and the carriage study was compared with registered invasive pneumococcal disease (IPD) data. This study is a part of the ProbiComp study, which was a double-blind, randomized controlled trial, including 290 children allocated to probiotics or placebo for 6 months and recruited during two autumn seasons (2014/2015). Pneumococci were identified by optochin sensitivity, bile solubility, α-hemolysis and/or capsular reaction. Serotyping was performed by latex agglutination kit and Quellung reaction. The carriage rate of S. pneumoniae was 26.0% at baseline and 67.4% at the end of intervention. No significant difference was observed between the placebo group and the probiotics group (p = 0.508). Children aged 8–19 months were carriers of non-pneumococcal vaccine serotypes causing IPD in children aged 0–4 years. However, serotypes causing most IPD cases in Danish elderly were either not found or found with low prevalence suggesting that children are not the main reservoir of those serotypes and other age groups need to be considered as carriers.

Introduction: Streptococcus pneumoniae in asymptomatic manner colonize the mucous membranes of the nasopharynx of children and adults, but can cause serious illness in the media which are naturally sterile. In 5-40% of healthy population this bacteria colonize the nasopharyngeal mucosa thanks to the surface adhesin protein, which allow the bacteria to attach to the epithelial cells. The normal nasopharyngeal microflora retains pneumococcus in a small number and does not allow it to express its pathogenic potential and cause disease. If this dominance of the normal microflora is violated, after adherence and local duplication, pneumococcus can spread to the middle ear, sinuses or lungs. Colonization is more common in children than in adults. Goal: The goal of this study was to determine the prevalence of the carrier state and susceptibility of pneumococcal strains that circulate in the outpatient population of Sarajevo Canton as a potential source of infection. Material and methods: In the microbiological laboratory of the Institute of Public Health of Canton Sarajevo in the period from July 1, 2013 until April 15, 2014 were analyzed swabs of the nose and nasopharynx, eye and ear from a total of 4109 outpatients. Swabs were inoculated on blood agar nutrient medium. Then was performed catalase test, preparation by Gram and susceptibility test on Optochin. Isolates positive for S. pneumoniae were subjected to in vitro assays to investigate the antimicrobial susceptibility/resistance. Results: Out of 4109 analyzed swabs the pneumococcus positive was 180 (4.38%). Of these, 137 (76.11%) nasal and nasopharyngeal swabs, 33 (18.33%) of the eyes and 10 (5.56%) ear. The highest number of positive swabs were isolated in children aged 6 years and less, a total of 168 (93.33%), in children aged 7-13 years were positive 7 (3.89%), while among respondents aged 14-20 years only 5 (2.78%). Conclusions: The most common site for isolation of pneumococci is the nose and throat, and the most common carriers of these bacteria are children under 6 years of age. Determining the prevalence of the carrier state contributes to the improvement of preventive measures to reduce the risk of infection and possible sequels.

Twenty six isolates of Streptococcus mitis bacteria collected from clinical of dental disease in missan city , then analyzed between (October and December in 2014).The samples taken in order to determine the bacterial profile and antibiotic susceptibility . Isolates of Streptococcus mitis were tested against eight different antibiotics and crude of crude aqueous extracts from flowers of Matricaria chamomilla L., by a disk diffusion method. 100% of isolates were resistat to the Tetracycline ,84%resistant to Ampicillin , 96% resistant Nitrofurantion ,100% resistant to Optochin ,80% resistant to Bacitracin ,96% resistant to, 92% resistant to Vincomycin , 73% resistant to Benzypenicillin where found to be the least effective antibiotics against MDR Streptococcus mitis. Also the antimicrobial suscepitility of isolates where tested against crude of crude aqueous extracts from flowers of Matricaria chamomilla L. with different concentration (62,5, 125, 250 , 500 mg / ml) detected by agar well diffusion methods , the aqueous extracts from flowers of Matricaria chamomilla L. have more effective in 500 mg / ml . Finally cytotoxicity evaluating toward human RBC , the results revealed these crud extracts of Matricaria chamomilla L. have not any cytotoxicity in all concentration . The good antimicrobial potency of crud extracts of Matricaria chamomilla L. indicates the treatment of MDR Streptococcus mitis as an alternative to the costly antibiotic.

Streptococcus pneumoniae is a major human pathogen that can survive to stress conditions, such as the acidic environment of inflammatory foci, and tolerates lethal pH through a mechanism known as the acid tolerance response. We previously described that S. pneumoniae activates acidic-stress induced lysis in response to acidified environments, favoring the release of cell wall compounds, DNA and virulence factors. Here, we demonstrate that F0F1-ATPase is involved in the response to acidic stress. Chemical inhibitors (DCCD, optochin) of this proton pump repressed the ATR induction, but caused an increased ASIL. Confirming these findings, mutants of the subunit c of this enzyme showed the same phenotypes as inhibitors. Importantly, we demonstrated that F0F1-ATPase and ATR are necessary for the intracellular survival of the pneumococcus in macrophages. Alternatively, a screening of two-component system (TCS) mutants showed that ATR and survival in pneumocytes were controlled in contrasting ways by ComDE and CiaRH, which had been involved in the ASIL mechanism. Briefly, CiaRH was essential for ATR (ComE represses activation) whereas ComE was necessary for ASIL (CiaRH protects against induction). They did not regulate F0F1-ATPase expression, but control LytA expression on the pneumococcal surface. These results suggest that both TCSs and F0F1-ATPase control a stress response and decide between a survival or a suicide mechanism by independent pathways, either in vitro or in pneumocyte cultures. This biological model contributes to the current knowledge about bacterial response under stress conditions in host tissues, where pathogens need to survive in order to establish infections. Fil: Cortes, Paulo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Piñas, German Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Cian, Melina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Yandar Barahona, Nubia Yadira. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Echenique, Jose Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Identification of Streptococcus pneumoniae among other α-haemolytic streptococci is based on phenotypic or genotypic characteristics such as colony morphology, bile solubility and optochin susceptibility. This study reports three optochin-resistant S. pneumoniae strains isolated from immunocompromised patients in Tunisia. The three isolates were positive for the bile solubility test. Biochemical identification with API® 20 Strep was not discriminatory for two strains. The three strains had different serotypes (6C, 19F and 23F) and three different sequence types (ST386, ST320 and ST326). Sequencing of the atpA and atpC genes for each strain showed only modification in atpC. The mutations Met13→Val or Val48→Ile were observed in two strains. However, in the third strain a novel type of mutation (Val15→Ile) was identified.

Despite the benefits of MALDI-TOF MS technology (Matrix-Assisted Laser Desorption-Ionization Time-Of-Flight Mass Spectrometry) reported worldwide and the continuous improving of the available databases, discrimination between Streptococcus pneumoniae and closely related species within the Streptococcus mitis group (SMG) using this methodology has been suboptimal. However, the accurate identification at the species level of this group of microorganisms is important for the appropriate management of infected patients. In this study, 216 SMG isolates -101 S. pneumoniae and 115 corresponding to 7 non-pneumococcal species within this group- were analyzed. All the isolates had been previously identified by conventional methods (optochin and bile solubility tests) and non-pneumococcal isolates were confirmed by sequence analysis (sodA and plys genes) when required. The isolates were also identified with the MALDI Biotyper 3.1 (Bruker Daltonics, Bremen, Germany) using an updated library containing 6,903 Main Spectra Profiles (MSPs). All the analyzed S. pneumoniae were correctly identified with MALDI-TOF MS at species level using the most updated database and all the non-pneumococcal SMG isolates were also identified at the group level. Several peaks (4,964.32, 6,888.90, and 9,516.46 m/z) have been found to be specific of S. pneumoniae, whilst a different set of peaks have proved to be present only in S. mitis (6,839.07 m/z) and S. oralis (5,297.61, 5822.53, and 6,839.07 m/z). Peak analysis allowed correct species assignment of 101/101 S. pneumoniae isolates (100%) and 102/105 S. mitis/oralis isolates (97.1%). Thus, the implementation of MALDI-TOF MS plus peak analysis for the identification of this group of microorganisms may provide precise species-level information that will allow the early implementation of directed antibiotic therapy.

Accurate identification of viridans group streptococci (VGS) frequently encountered as a causative agent of infective endocarditis is always a challenge for the clinical microbiology laboratory. Clinical microbiology laboratories generally use semi automatic/full automatic systems, molecular methods and also conventional methods for the identification of these bacteria. There are recent published studies that have used MALDI-TOF (Matrix Assisted Laser Ionization Mass Spectrometry-Time of Flight) systems in the identification of VGS. The aim of the study was to compare the performance of the conventional methods, semi automatic and MALDI-TOF MS system used in identification of VGS in oral microbiota of persons under the risk of infective endocarditis, with the gold standard method 16S rRNA sequence analysis and to create a diagnosis algorithm for the identification of VGS in clinical microbiology laboratories according to the obtained data. The study was conducted with 51 VGS strains isolated from oral microbiota of the patients with rheumatologic cardiac, valve and/or prosthetic valve diseases, under the risk of development of infective endocarditis, who have admitted to Ankara Numune Training and Research Hospital, Department of Cardiology, between February-June 2015. Standard microbiology procedures, optochin susceptibility and bile solubility tests were done for the isolation of bacteria. Bacteria were also identified with APISTREP (bioMerieux, France) and MALDI-TOF MS Bruker Microflex (Bruker Biotyper; Bruker Daltonics, Bremen, Germany) methods. BSF-8 (5'-AGAGTTTGATCCTGGCTCAG-3') and BSR-534(5'-ATTACCGCGGCTGCTGGC-3') primers were used in the 16S rRNA sequence analysis of bacteria. ABI PRISM 3100 Avan t Genetic Analyzer (Applied Biossytems, Foster City, CA, USA) were used for the sequence analysis. Electropherograms were analyzed in SeqScape Software (Applied Biosystems, Foster City, CA, USA) and compared with the reference sequences in GenBank with BLASTN (NCBI). According to the result of optochin and bile solubility tests, with API STREP system, 16 (31,37%) of the isolates were identified as Mitis group, 15 (29.41%) as Anginosus group, 9 (17.5%) as Salivarius group, 7 (13,73%) as Sanguinis group and 4 (7.84%) as Bovis group among optochin and bile resistant alpha hemolytic streptococci. Moreover, of the same isolates 20 (39.22%) were identified as Mitis group, 14 (27.45%) as Anginosus group, 13 (25.49%) as Salivarius group and 4 (7.84%) as Sanguinis group with MALDI-TOF system. In the identification with 16S rRNA, 25 (49.02%) of the isolates were identified as Mitis group, 13 (25.49%) as Anginosus group, 12 (23.53%) as Salivarius group and 1 (1.96%) as Sanguinis group. According to the results, it was determined that 33 (64.70%) of the isolates identified in MALDI-TOF MS system and 31 (60.78%) of the isolates identified in API STREP system were compatible with 16S rRNA sequence analysis method. For Mitis group, API STREP test sensitivity was 48.00% and specificity was 84.62% and MALDI-TOF system sensitivity was 80.00% and specificity was 100%. As VGS identification is a complicated process, we believe a single method will be insufficient for the identification of these isolates in clinical microbiology laboratories. We suggest that MALDI-TOF system can be used for VGS diagnosis, however, optochin test and/or molecular methods should also be included in the diagnosis algorithm when necessary.

The taxonomy of streptococci has undergone major changes during the last two decades. The present classification is based on both phenotypic and genotypic data. Phylogenetic classification of streptococci is based on 16S rRNA sequences [1], and it forms the backbone of the overall classification system of streptococci. Phenotypic properties are also important, especially for clinical microbiologists. The type of hemolysis on blood agar, reaction with Lancefield grouping antisera, resistance to optochin, and bile solubility remain important for grouping of clinical Streptococcus isolates and therefore treatment options [2]. In the following chapter, two phenotypic classification groups, viridans group streptococci (VGS) and beta-hemolytic streptococci, will be discussed.

Background The aim of the present study was to investigate the genetic relatedness and the antimicrobial resistance profiles of a collection of Austrian Streptococcus pneumoniae isolates from companion animals and horses. A total of 12 non-repetitive isolates presumptively identified as S. pneumoniae were obtained during routinely diagnostic activities between March 2009 and January 2017. Results Isolates were confirmed as S. pneumoniae by bile solubility and optochin susceptibility testing, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry and sequence analysis of a part recA and the 16S rRNA genes. Isolates were further characterized by pneumolysin polymerase chain reaction (PCR) and genotyped by multilocus sequence typing (MLST). Antimicrobial susceptibility testing was performed and resistance genes were detected by specific PCR assays. All isolates were serotyped. Four sequence types (ST) (ST36, ST3546, ST6934 and ST6937) and four serotypes (3, 19A, 19F and 23F) were detected. Two isolates from twelve displayed a multidrug-resistance pheno- and genotype. Conclusions This study represents the first comprehensive investigation on characteristics of S. pneumoniae isolates recovered from Austrian companion animals and horses. The obtained results indicate that common human sero- (23F) and sequence type (ST36) implicated in causing invasive pneumococcal disease (IPD) may circulate in dogs. Isolates obtained from other examined animals seem to be host-adapted.

Streptococcus pseudopneumoniae is a distinct pathogen and the available evidence suggests its significance in lower respiratory tract infections. The purpose of this study is to investigate the occurrence of this new species in patients presenting with community-acquired pneumonia in a university hospital setting using routine laboratory tests. Sputum samples (n = 213) submitted to the microbiology laboratory for culture from cases clinically diagnosed as community-acquired pneumonia are studied. Optochin variability in ambient air and 5% CO2 was the criterion used to identify S. pseudopneumoniae. Disc-diffusion and E-test on Muller Hinton blood-based agar were used for antibiotic susceptibility testing following Clinical Laboratory Standard Institute breakpoints. Out of the screened isolates, only six (3%) were identified as S. pseudopneumoniae. None of the commercial tests differentiated the pathogens from viridans streptococci so diagnostic laboratories need to provide an alternative identification algorithm. The six isolates showed susceptibility to penicillin, and five isolates were susceptible to vancomycin, but one exhibited low-level resistance (minimum inhibitory concentration [MIC]: 1 μg/mL). All isolates showed high-level resistance to macrolides (MIC: 24- > 256 μg/mL). Although S. pseudopneumoniae strains were not sufficiently frequent to justify routine additional incubation of sputum samples in ambient air, the high resistance pattern is alarming. Further surveillance is needed in diagnostic laboratories to understand the epidemiology and clinical significance of the newly described pathogen.

BackgroundPneumonia caused by Streptococcus pneumoniae is the leading cause of death among children in Ethiopia. S. pneumoniae nasopharyngeal carriage can result in endogenous infections and bacterial spread in the community. S. pneumoniae drug resistance is rapidly increasing worldwide. The aim of the study was to assess the nasopharyngeal carriage and antimicrobial susceptibility pattern of S. pneumoniae among pediatric outpatients.MethodsA cross-sectional study was conducted on children aged ≤10 years from February to May of 2012. Data on potential risk factors were gathered using an interview-based questionnaire. Nasopharyngeal specimens were collected with a sterile plastic cotton tip swab. Bacteria were characterized by colony appearance, Gram staining, and optochin susceptibility and bile solubility tests. An antimicrobial susceptibility test was performed using the disk diffusion method. A logistic regression analysis was used to examine the possible risk factors. All tests with p value less than 0.05 were considered statistically significant.ResultsOf the 234 children screened, 41.03% carried S. pneumoniae. Age-related downward trend for S. pneumoniae carriages (50.9% in

Butanol-resistant bacteria were isolated from butanol solvent. The cell growth of isolated strains declined with increasing concentrations of butanol, and isolated strain BRS02 displayed more resistance to 12.5 g/L of butanol than other isolated strains. In addition, strain BRS251, which was resistant to even higher concentrations of butanol, was developed by the mutation of BRS02 using UV. BRS251 could grow in LB medium containing up to 17.5 g/L of butanol, 32.5 g/L of propanol, or 6 g/L of pentanol, whereas the control strain Escherichia coli was found to be tolerant to 7.5 g/L of butanol, 20 g/L of propanol, or 2 g/L of pentanol. The isolated BRS02, a Gram(+) bacterium seen to have a cocci form under the microscope, grew in 6.5% NaCl. According to biochemical tests, BRS02 can metabolize and produce acid with D-galactose, D-maltose, D-mannitol, D-mannose, methyl--Dglucopyranoside, D-ribose, sucrose, or D-trehalose, as carbon sources. Also, this strain showed resistance to bacitracin, vibriostatic agent O/129, and optochin, alongside positive activities for arginine dihydrolase, -glucosidase, and urease. The BRS02 strain was identified as Staphylococcus sp. by analyses of the 16S rRNA gene, phylogenetic tree, and biochemical tests.

INTRODUCTION: S.pneumoniae is the major cause of morbidity and mortality in India and abroad and carriage is the key to invasive disease. Carriage (20-60%) and invasive disease are more prevalent in children and in the elderly. Increased case fatality is due to the emergence of Penicillin and Multidrug Resistant S.pneumoniae worldwide. Penicillin has been the drug of choice for treatment of pneumococcal infections but the increasing number of reports of penicillin resistant pneumococci (PRP) throughout the world makes it essential to regionally determine the prevalence of PRP. MATERIALS AND METHODS: Nasopharyngeal swabs were collected from children between ≥ 3 months and ≤ 5 years of age attending paediatric immunization OPD in a medical college tertiary care hospital and research centre, Bengaluru, between December 2008 and August 2009 ( 8 months). S. pneumoniae were isolated based on colony morphology, Gram's staining, optochin sensitivity, and bile solubility. Antimicrobial susceptibility testing was carried out by Kriby-Bauer disc diffusion method for recommended commonly prescribed antibiotics. Oxacillin resistant strains denoting penicillin resistance were tested for Minimum inhibitory concentration for Penicillin G by Epsilometer test. RESULTS: 53 (27.89 %) S. pneumoniae were isolated from 190 nasopharyngeal swabs. 9 (16.98%) isolates have been intermediately penicillin resistant by E test. Only 5 (9.43%) and 34 (64.15%) isolates were sensitive to co-trimoxazole and tetracycline respectively. 51 isolates were susceptible to cefotaxime, 48 (90.56%) each were susceptible to erythromycin and ciprofloxacin. 25(47.19%) out of 53 isolates could be recovered at the end of one year stored in STGG medium at -20˚ C. Serogroup types 19, 10, 3, 14, 7, were common among the isolates. CONCLUSION: Carriage of S. pneumoniae is prevalent among young children in Bengaluru, which may predispose them to subsequent invasive pneumococcal diseases. There is decreased susceptibility to penicillin and other recommended antibiotics that may warrant appropriate culture and sensitivity testing during the management of invasive pneumococcal diseases and prescription of antibiotics in general.

Fourteen fluoroquinolone-resistant streptococcal isolates with recombinant DNA topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. Phenotypic tests classified them as atypical pneumococci. Phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of Streptococcus pneumoniae , Streptococcus mitis , Streptococcus oralis , and Streptococcus pseudopneumoniae . Four isolates grouped with S. pneumoniae , seven grouped with S. pseudopneumoniae , and three grouped with S. mitis . These results generally agreed with those obtained with an optochin susceptibility test and with the organization of the atp operon chromosomal region, encoding the F o F 1 H + -ATPase (the target of optochin). All seven isolates grouping with S. pseudopneumoniae share the same spr1368-atpC-atpA gene order; all four grouping with S. pneumoniae share the spr1368 -IS 1239-atpC-atpA order, and two out of the three grouping with S. mitis share the spr1284-atpC-atpA order. In addition, evidence for recombination within the seven housekeeping alleles of the S. pseudopneumoniae population was provided by several methods: the index of association (0.4598, P < 0.001), the pairwise homoplasy index, and the split-decomposition method. This study confirms the existence of pneumococci among the alpha-hemolytic streptococci with DNA topoisomerase genes showing a mosaic structure and reveals a close relationship between atypical pneumococci and S. pseudopneumoniae .

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae , have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae , 2 S. pseudopneumoniae , and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO 2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O 2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.

Accurate species-level identification of viridans group streptococci (VGS) is very important for understanding of their pathogenicity and virulence. However, an extremely high level of the similarity between VGS, especially Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis and Streptococcus pseudopneumoniae, often results in misidentification of these organisms, so there is an urgent need of novel approaches to species identification. A set of 50 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (alpha-hemolysis, colony morphology, Gram stain and optochin susceptibility). Modern proteomic and genetic approaches - the direct bacterial profiling (DBP) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and multilocus sequence analysis (MLSA) scheme (http://viridans.emlsa.net/) - were applied for the accurate species identification. After that all isolates were stored at -70°C. Later they were re-inoculated, and a number of additional tests (bile solubility, latex agglutination by commercial "Slidex® pneumo-kit" and repeated optochin test) were performed. A considerable discrepancy was discovered in the results of the different approaches. Looking in the future, one could say that MLSA-like schemes based on the analysis of the nucleotide sequences of seven or more loci of the bacterial genome, appeared to be the most useful instrument in the VGS discrimination, in contrast to the numerous one-target identification schemes, which have been introduced into practice by now.

Background: Streptococcus species account for 17%–50% of the microorganisms causing infectious endocarditis (IE), but S. pneumoniae rarely causes IE. Most clinical microbiology laboratories today depend on the optochin test to identify pneumococci. However, there have been several reports of optochin-resistant pneumococci. Case Report: A 64-year-old man suffered from sepsis and had a large vegetation on the aortic valve. The bacteria detected in the blood cultures showed α-hemolysis and were optochin-resistant, which suggested that these microorganisms were viridans streptococci. However, the bile solubility test, additional biochemical testing, and genetic analysis by polymerase chain reaction (PCR) revealed that the isolate was S. pneumoniae . Conclusions: Alpha-hemolytic streptococci showing optochin resistance should undergo additional tests before being identified as viridans streptococci, especially if the strain has been isolated from a sterile site in the body, such as the blood or cerebrospinal fluid. * IE : infectious endocarditis PCR : polymerase chain reaction AST : aspartate transaminase ALT : alanine transaminase LDH : lactate dehydrogenase HN : medical technologist lytA : autolysis gene ply : pneumolysin gene MIC : minimal inhibitory concentration CLSI : Clinical Laboratory Standards Institute

Objective: The aim of this study was to establish cases of bacteria associated with lower respiratory tract infections (LRTIs) in General Hospital Dr. Pirngadi Medan with the view to identify the bacteria well as to assess their antibiotics susceptibility. Methods: An observation and prospective study were analyzed to patients with lower respiratory tract infection in respiratory and internal wards Dr. Pirngadi Hospital Medan. For the identification method of bacteria was using optochin test, catalase test, Mannitol salt agar, coagulase, indole, methyl red, Voges–Proskauer test, citrate, urease, and Triple Sugar Iron test. Results: The bacteria that cause lower respiratory tract infection were Pseudomonas aeruginosa (25.53%), Klebsiella pneumoniae (17.02%), Proteus sp. (14.89%), Staphylococcus aureus (12.77%), Escherichia coli (10.64%), Streptococcus pneumoniae (10.64%), and Haemophilus influenzae (8.51%). The result of sensitivity testing showed that gentamicin is more sensitive and the resistant antibiotic is chloramphenicol among all the tested antibiotics. Conclusion: P. aeruginosa was the most common isolated from LRTIs patients with gentamicin is the most sensitive antibiotics while chloramphenicol is the most resistant antibiotics among all the tested antibiotics.

Background: Infections with Streptococcus pneumoniae are endemic worldwide. It is a public health problem and responsible for 1.6 million of 8.8 million annual deaths of children under 5 years of age, with 50% occurring in sub-Saharan Africa. This descriptive study was done to determine the prevalent S. pneumoniae serotypes responsible for infections at Ahmadu Bello University Teaching Hospital, Shika-Zaria, Nigeria. Materials and Methods: Clinical specimens of blood, cerebrospinal fluid, and aspirates from abscess, ear swab, throat swab, pus and sputa were collected over a period of 18 months from 420 patients with pneumonia, meningitis, septicemia, and otitis media. Specimens were cultured on 5% defibrinated sheep blood agar and chocolate agar. Incubation was done aerobically in a CO2-enriched atmosphere at 37°C for 18–24 h. Isolates of S. pneumoniae were identified by standard biochemical techniques using Gram reaction, catalase test, Optochin disc, and bile solubility tests. Antimicrobial susceptibility was determined by the modified Kirby–Bauer disc diffusion method with Mueller–Hinton agar supplemented with 5% sheep blood. Serotyping was done using the slide agglutination method (Denka Seiken Co. Ltd., Japan). The serotype final results were recorded as matching, discordant, or nontypeable. Results: A total of 420 patients participated in this study, in which 227 were males (54%) and 193 were females (46%). Participants' ages ranged from 2 days to 85 years. S. pneumoniae isolates were mainly from blood 12 (52.2%) and sputum 6 (26.1%). Samples with most isolates were from the pediatric age group of 15 years (65.2%). The serotypes identified were 6, 19, and 20 which were all from blood, as none of the strains from sputum was typeable. Conclusion: The major S. pneumoniae serotypes found in this study were 6, 19, and 20.

Objectives To investigate the prevalence of Streptococcus pneumoniae (pneumococci) in the nasopharynx of healthy children enrolled in public day-care centers of the municipality of Umuarama, state of Parana, Brazil. The susceptibility of the pneumococcal strains to antimicrobial agents was also studied. Methods Nasopharyngeal specimens from 212 children were collected from April to October 2008. After the specimens were seeded in blood agar and incubated at 37 degrees C for 24-48 hours, the colonies suspected of belonging to S. pneumoniae were identified using alpha-hemolysis, optochin sensitivity, and bile solubility test. Penicillin susceptibility was investigated using the disk diffusion and dilution tests. Susceptibility to the other antimicrobial agents indicated for the treatment of pneumococcal infections was investigated using the disk diffusion test. Results The prevalence of nasopharyngeal pneumococci was 43.4% (92/212), with higher rates in children between 2 and 5 years old (p = 0.0005). There was no significant difference between sexes. Intermediate and full resistance to penicillin were found in 34.8 (32/92) and 22.8% (21/92) isolates, respectively. Sixty-seven strains (72.8%) were resistant to sulfamethoxazole-trimethoprim, eight (8.7%) were resistant to erythromycin, and six (6.5%) to tetracycline. One strain was resistant to clindamycin (1.1%) and another was resistant to chloramphenicol (1.1%). All strains were sensitive to levofloxacin, ofloxacin, rifampicin, telithromycin, linezolid, and vancomycin. Nine strains were considered multiresistant because they were resistant to three or more classes of antimicrobial agents. Conclusions The present study detected a high prevalence of healthy children colonized with penicillin-resistant S. pneumoniae strains who may be important reservoirs of this pathogen in the community.

OBJECTIVES: To investigate the prevalence of Streptococcus pneumoniae (pneumococci) in the nasopharynx of healthy children enrolled in public day-care centers of the municipality of Umuarama, state of Parana, Brazil. The susceptibility of the pneumococcal strains to antimicrobial agents was also studied. METHODS: Nasopharyngeal specimens from 212 children were collected from April to October 2008. After the specimens were seeded in blood agar and incubated at 37 °C for 24-48 hours, the colonies suspected of belonging to S. pneumoniae were identified using α-hemolysis, optochin sensitivity, and bile solubility test. Penicillin susceptibility was investigated using the disk diffusion and dilution tests. Susceptibility to the other antimicrobial agents indicated for the treatment of pneumococcal infections was investigated using the disk diffusion test. RESULTS: The prevalence of nasopharyngeal pneumococci was 43.4% (92/212), with higher rates in children between 2 and 5 years old (p = 0.0005). There was no significant difference between sexes. Intermediate and full resistance to penicillin were found in 34.8 (32/92) and 22.8% (21/92) isolates, respectively. Sixty-seven strains (72.8%) were resistant to sulfamethoxazole-trimethoprim, eight (8.7%) were resistant to erythromycin, and six (6.5%) to tetracycline. One strain was resistant to clindamycin (1.1%) and another was resistant to chloramphenicol (1.1%). All strains were sensitive to levofloxacin, ofloxacin, rifampicin, telithromycin, linezolid, and vancomycin. Nine strains were considered multiresistant because they were resistant to three or more classes of antimicrobial agents. CONCLUSIONS: The present study detected a high prevalence of healthy children colonized with penicillin-resistant S. pneumoniae strains who may be important reservoirs of this pathogen in the community.