Streptococcus pseudopneumoniae as an emerging pathogen from patients with respiratory diseases

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.


INTRODUCTION
Streptococcus pseudopneumoniae as suggested by DNA-DNA homology studies is recorded as a species of the Streptococcus mitis/oralis members of viridans group Streptococci; they have some similar features to Streptococcus pneumoniae (Arbique et al., 2004).The principle characteristics of S. pseudopneumoniae are the lacking of pneumococcal capsule, bile insolubility, indeterminate sensitivity or resistance to optochin after *Corresponding author.E-mail: nohat75@yahoo.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License incubation in CO 2 (5%), while sensitivity to it after incubation in ambient air, they give positive results with antigen detection and DNA probe hybridization tests (Keith et al., 2006).
S. pseudopneumoniae was firstly described in sputum samples of patients with lower respiratory tract infection, particularly those with Chronic Obstructive Pulmonary Diseases (COPD) (Laurens et al., 2012).The clinical relevance and pathogenic potential of this organism has not been clearly determined except in few studies that were conducted on mouse models with sepsis and peritonitis, so the correct identification and rapid detection of this emerging pathogen in clinical setting is very important for determination of its disease potential (Harf-Monteil et al., 2006).
S. pseudopneumoniae is usually misidentified in our laboratories as we usually rely on optochin sensitivity testing with 5% CO 2 for detection of S. pneumoniae and miss the incubation in ambient air which is the key point for identification of S. pseudopneumoniae.Biochemical reactions are usually used to differentiate typical S. pneumoniae from S. pseudopneumoniae and other related Streptococci; several molecular techniques have also been used (Rolo et al., 2013).
There are several polymerase chain reaction (PCR)based assays that target specific pneumococcal virulence factors for the typical S. pneumoniae, such as autolysin A (lytA) which is the major pneumococcal autolysin (Simo˜es et al., 2010) and the capsular polysaccharide biosynthesis gene A (cpsA) which is a conserved pneumococcal capsular polysaccharide gene (Bentley et al., 2006), also aliB-like ORF2 (a gene that usually found in the capsular region of non-capsulated pneumococci) (Hathaway et al., 2004); these genetic markers have been used to differentiate S. pneumoniae from other related species (Rolo et al., 2013).
Unknown putative genes, specific intergenic DNA sequences, or specific regions of the 16S rRNA, have also been proposed to be pneumococcal species-specific (El Aila et al., 2010).
To the best of our knowledge, there are no published studies on the isolation of S. pseudopneumoniae from pathological specimens in Egypt.So the aims of the present study were to isolate S. pseudopneumoniae from sputum samples of patients admitted to Chest Department of Mansoura University Hospitals (MUHs) and to differentiate it from S. pneumoniae and other viridans group Streptococci by phenotypic and genotypic tests as well as to determine the prevalence of this overlooked pathogen and the risk factors of its acquisition.

Design of the study
Cross sectional descriptive study was conducted on 381 patients El-Kazzaz et al. 1339 showing signs of respiratory tract infection (Horan et al., 2008) during period extending from the first of September, 2015 to the end of August, 2016.All patients, enrolled in this study, were admitted to the Chest Department of MUHs.The protocol of this research was approved by the ethical committee in the Faculty of Medicine, Mansoura University, with code number R/16.10.08.

Sample collection
Sputum samples were collected from all studied patients (one sample from each patient) under complete aseptic condition.The samples were processed in the Unit of Microbiology Diagnostic and Infection Control in the Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University.

Microscopic examination
Prepared films stained with Gram from sputum specimens were microscopically examined.Specimens with >25 leukocytes and <10 squamous epithelial cells/×100 microscopic field were incorporated in this study and subjected to advance processing, whereas specimens with >10 epithelial cells/×100 microscopic field were not included in the research (Goyal et al., 2007).
After microscopic examination of the collected 381 sputum samples, 329 samples fulfilling aforementioned criteria were subjected to further processing and 52 samples were excluded.

Culture and identification
The 329 sputum specimens were cultivated on 5% sheep blood agar, chocolate agar (Oxoid, UK), and MacConkey's agar (HiMedia Laboratories, India) plates by taking a loopful from the purulent portion of each sputum specimen using calibrated bacteriological loop that carry 0.01 ml.Cultivation on blood agar and chocolate agar plates were done in duplicate; one was incubated in CO2 enriched atmosphere using a candle jar for 24 h at 37°C and the other was incubated aerobically at 37°C for 24 h.MacConkey's agar plates were incubated aerobically at 37°C for 24 h (Zhou et al., 2011).Significant count of bacteria has been determined as being ≥10 5 CFU/ml, whereas bacteria < 10 5 CFU/ml was considered to be non-significant count of normal flora (Amissah and Pappoe, 2014).Isolated organisms were identified according to standard microbiological methods.Alpha hemolytic colonies were selected for Gram staining, colonies that showed Gram positive cocci arranged in pairs or chains were subjected to further identification by pneumococcal capsule detection test (capsules were detected by observing a halo around Pneumococci with India ink at ×400 magnification) (Harf-Monteil et al., 2006), optochin susceptibility and bile solubility tests.

Optochin susceptibility test
The test was done in duplicate; one plate was incubated in 5% CO2 and the other in ambient air (O2 atmosphere) at 35°C for 18 to 24 h.Inhibition zones around the disks were measured and interpreted according to the recommended standards of CLSI (2014).Positive test showed a zone of inhibition of 14 mm or more around the disk (Koneman et al., 2006).

Bile solubility test
The test was conducted with bile solubility reagent (10%) (Remel, USA).Clearing of the test suspension within 3 h was considered to be positive test (Wessels et al., 2012).All S. pseudopneumoniae isolates included in this study were phonotypically suspected as being capsule free, sensitive to optochin in ambient air but intermediate or resistant by incubation in 5% CO2 and bile insoluble.

Maintenance of the selected alpha hemolytic Streptococci
For isolates preservation, skim-milk tryptone glucose glycerol (STGG) medium was used (O'Brien et al., 2001).Pure growth of each isolate was collected and dispensed into 1.5 ml vials containing 1.0 ml of STGG medium.After that, the suspension was stored at -70°C for further testing.Before starting any further experiment, subculture of the isolates on blood agar was done twice to allow isolates to restore their viability (Charalambous et al., 2003).

Design of specific gene primers for multiplex PCR assay
A multiplex PCR was performed for all of studied alpha hemolytic Streptococci in order to detect internal fragments of the following four genes: cpsA, lytA, aliB-like ORF2 and 16S rRNA gene as a positive internal control (Rolo et al., 2013).The primer sequences and expected band sizes have been used in accordance with previous studies (Table 1).

DNA extraction
DNA was extracted using QIAamp® DNA Mini kits, QIAGEN (Germany) according to the manufacturer's instructions.

Multiplex PCR technique
The multiplex PCR was done in a 10 µl volume with 1×PCR buffer (10 mM Tris-HCl, pH 8.3/50 mM KCl/1.5 mM MgCl2), 0.12 mM of dNTPs Mix (2 mM each), 1 U/µl of Taq DNA polymerase (Fermentas) and 0.5 pmol/µl of each primer (Simo˜es et al., 2011). 1 µl of extracted DNA was added as DNA template.PCR was done with the following reaction conditions: a pre-denaturation step of 4 min at 94°C; 35 cycles at 94°C for 30 s, 52°C for 45 s, and 72°C for 1 min; a final extension step of 5 min at 72°C; and soaking at 16°C.The PCR products were analyzed after running on 2% agarose gel containing ethidium bromide at 80 volts for about 2 h on comparison to DNA standard marker: #SMO373, 50 bp DNA Ladder, Thermo Scientific Inc., USA (Richter et al., 2013;Hajia et al., 2014).Distilled water was incorporated in every run of the multiplex PCR as a negative control.

Testing for antimicrobial sensitivity
S. pseudopneumoniae isolates were subjected to antibiotic sensitivity testing as recorded by the recommendations of CLSI using disk diffusion method on Muller-Hinton agar (Oxoid, UK) supplemented with 5% sheep blood using the following antibiotics: penicillin, amoxicilline/clavulinic acid, erythromycin, clindamycin, cefotaxime, co-trimoxazole, ciprofloxacin, linezolid, cefepime, vancomycin, meropenem and teicoplanin (Forbes et al., 2007).The inhibitory zone limits of the tested antimicrobials were referred to CLSI (2014) guidelines.

Analysis of data
The data were entered and analyzed statistically with Statistical Package of Social Science (SPSS) using software version 21 (SPSS Inc., Chicago, IL, USA).Descriptive statistics were described as mean, standard deviation (SD), minimum, maximum and percentage.Inter-group comparison of categorical data was done using Chi square test (2-value).Odds ratio (OR) and confidence interval (CI) were used to calculate the risk factors.For all of the previous statistical tests, the threshold of significance is fixed at 5% (p-value <0.05 was considered to be statistically significant).

RESULTS
Out of the studied 329 sputum samples, 286 yielded single pathogen whereas 13 yielded more than one pathogen (2 pathogens) and mixed insignificant growth of normal respiratory tract flora was detected in the remaining 30 samples.The total number of the isolated respiratory pathogens was 312.Sixty two isolates (62/312, 19.9%) were alpha hemolytic Streptococci whereas the remaining 250 isolates were Klebsiella Among the isolated 62 alpha hemolytic Streptococci, 16 isolates were presumptively recognized as being S. pseudopneumoniae by phenotypic tests, as all these isolates lacked the pneumococcal capsules, showed resistance to optochin (zone diameter <14 mm) in 5% CO 2 incubation, but they were sensitive to optochin (zone diameter between 18 to 25 mm) when incubated in ambient air; also they were found to be bile insoluble.Regarding the colony morphology of the S. pseudopneumoniae on 5% sheep blood agar, they were observed to be small (about 1 mm in diameter), shiny, smooth, and domed, with entire edges.Few colonies having depressed centers, seem to be a smaller version of the S. pneumoniae draftsmen colonies.The remaining 46 alpha hemolytic Streptococci were identified as S. pneumoniae (21 isolates) and other viridans group Streptococci (25 isolates) accounted for 6.7 (21/312) and 8.0% (25/312) of the isolated respiratory pathogens, respectively.The percentage of the isolated S. pseudopneumoniae in relation to other detected respiratory pathogens was 5.1% (16/312) and its prevalence among studied patients was 4.9% (16/329).
The multiplex PCR assay which was conducted on the studied alpha hemolytic Streptococci showed that DNA from 2/16 of the putative S. pseudopneumoniae isolates and 5/25 of the viridans group Streptococci were negative for 16S rRNA gene, so these extracts must have been inhibitory.S. pseudopneumoniae PCR results for the other targets therefore were determined out of the 14 (non-inhibitory) samples.All of the phenotypically identified S. pseudopneumoniae isolates were negative for cpsA and lytA genes, but they were positive for aliBlike ORF2 (Table 2 and Figure 1).
Antibiotic sensitivity testing of the studied 16 S. pseudopneumoniae isolates showed that all of them were sensitive to linezolid, vancomycin and teicoplanin, on the other hand, co-trimoxazole, penicillin and erythromycin recorded the lowest sensitivity for those isolates (Table 3).
The distinctive data of patients with culture positive sputum for S. pseudopneumoniae (16 patients) showed that their mean age was 58.06 years (range 17-76 years); 56.2% were males, 68.8% were presented with COPD with infected exacerbation.On the other hand, only 37.5% of them showed positive X-ray for infiltration and 31.2%recorded elevated leukocyte counts (>11 × 10 9 cells/L).S. pseudopneumoniae was the only detected pathogen in 68.8% of sputum cultures of those patients, whereas H. influenza was the most common co-pathogen detected in sputum culture of patients that showed other bacterial growth along with S. pseudopneumoniae (Table 4).
On comparing patients with culture positive sputum for S. pseudopneumoniae with patients who showed the carriage of other alpha hemolytic Streptococci (S. pneumoniae and viridans group Streptococci) in their sputum samples, COPD with infected exacerbation and previous hospitalization with COPD were found to be more significantly associated with the acquisition of S. pseudopneumoniae infections (P= 0.006 and 0.01, respectively).On the other hand, there was no statistically significant difference regarding presence of asthma, pneumonia, bronchiectasis, bronchogenic carcinoma or smoking history as risk factors (Table 5).

DISCUSSION
S. pseudopneumoniae may often be overlooked during examination of sputum samples of patients with respiratory disorders particularly in our locality.Few studies have been conducted on the isolation and description of this organism; the prevalence of S. pseudopneumoniae is determined in few researches that have been considered to be unique for certain areas of the world and this research was one of the first attempts to isolate this organism from patients with respiratory diseases in our locality.
S. pseudopneumoniae could be easily isolated and characterized by few phenotypic and genotypic tests  The prevalence of S. pseudopneumoniae among studied patients with respiratory tract disorders was 4.9%; this percentage approximates what has been recorded in previous studies at which this pathogen was isolated from nearly 4% of the studied cases (Keith et al., 2006;Mohammadi and Dhanashree, 2012).On the other hand, lower percentage of S. pseudopneumoniae (nearly 1%) has been observed by other researchers (Harf-Monteil et al., 2006;Swathi et al., 2015).The variability of results among various studies may be due to the Checking for specific genetic markers by multiplex PCR revealed the absence of cpsA and lytA genes in all studied isolates of S. pseudopneumoniae, whereas they were found to be, respectively positive in 90.5 and 100% of the isolated S. pneumoniae.These findings support a previous published study at which cpsA was confirmed to be a specific capsular polysaccharide gene exclusively harbored by S. pneumoniae as it is commonly linked with the capsular operon and absent in S. pseudopneumoniae isolates as being non capsulated (Park et al., 2010).Similarly, lytA gene which has been considered to be one of the diagnostic markers that differentiate S. pneumoniae from other closely related species including S. pseudopneumoniae was also found to be absent in all isolates of S. pseudopneumoniae that have been examined in other researches (Rolo et al., 2013).
aliB-like ORF2 gene was detected in all studied S. pseudopneumoniae isolates supporting the observations recorded in previous researches that have mentioned this gene as being specific for non-capsulated Pneumococci and hence S. pseudopneumoniae as being non capsulated (Hathaway et al., 2004).There were three isolates of the examined S. pneumoniae found to be positive for aliB-like ORF2 gene; our results are in agreement with previous findings that have recorded the same observation (Simo˜es et al., 2011) which could be explained by the lack of ability of those isolates to produce the capsule in vitro (Scott et al., 2012) as a result of capsular genes mutation (Melchiorre et al., 2012).
A great resistance rate of the studied S. pseudopneumoniae isolates was recorded for cotrimoxazole (75.0%), penicillin (62.5%) and erythromycin (56.2%) which was consistent with previous studies at which the same resistance pattern has been observed among studied isolates (Keith and Murdoch, 2008;Mohammadi and Dhanashree, 2012).Other studies have also documented a high resistance rate among S. pseudopneumoniae isolates to erythromycin (60%) without any recorded resistance to penicillin (Keith et al., 2006) that was in contrast with our finding and may be due to different antibiotic policy in other countries at which penicillin is used within limits.Unfortunately, there is no recorded information about isolation and antibiotic sensitivity profile of S. pseudopneumoniae in our locality to be compared with our findings.The observed high resistance of this organism to penicillin and other classes of antibiotics highlights its role as one of the respiratory tract pathogen that should not be neglected particularly during selection of the appropriate antibiotic that could be used in dealing with respiratory tract infection.
In this study, S. pseudopneumoniae was the only pathogen recovered from 11/299 (3.7%) of specimens that were positive both by smear-microscopy and culture for bacterial pathogens and it was the predominant one in samples that showed growth of other co pathogen.This observation should not be neglected as it may be an indirect evidence of the important clinical role of this pathogen in the acquisition of respiratory tract infection.This observation was also supported by the presence of pulmonary infiltrate in 37.5% and elevated leukocyte count in 31.2% of cases with culture positive S. pseudopneumoniae.Although a high resistance rate of S. pseudopneumoniae to various classes of antibiotics has been documented in previous studies, most of them suspected this organism as a colonizing bacterium of the respiratory tract or a weak opportunistic pathogen that is usually associated with potentially pathogenic organisms in patients with chronic conditions (Laurens et al., 2012).Subsequent studies have supported our finding that this organism may have a real clinical importance depending on the observed high resistance rate for this organism together with its involvement in various types of invasive diseases (Rolo et al., 2013).
In this study, the actual risk factors associated with S. pseudopneumoniae infection were assessed by comparing patients with culture positive sputum for S. pseudopneumoniae with patients who revealed other alpha hemolytic Streptococci (S. pneumoniae and viridans group Streptococci) in their sputum cultures.COPD with infected exacerbation and previous hospitalization with COPD were significantly associated with increase in the acquisition of S. pseudopneumoniae in spite of large number of studied cases suffering from COPD in this research.The association of COPD with S. pseudopneumoniae infection has been documented by previous studies (Keith et al., 2006;Harf-Monteil et al., 2006), however other studies have recorded that S. pseudopneumoniae were more frequently associated with bronchitis and pneumonia rather than COPD (Laurens et al., 2012).To determine the actual role of S. pseudopneumoniae in COPD exacerbation, cohort studies for patients during periods of exacerbation and stability of COPD is recommended to observe the appearance of new S. pseudopneumoniae isolates in the exacerbation periods that could confirm the association of infection with new bacterial strains and COPD exacerbation (Sethi et al., 2002).
Future studies seems to be necessary to determine the actual habitat, epidemiology, colonization of S. pseudopneumoniae and if it is associated with respiratory diseases other than COPD particularly pneumonia.

Conclusion
The pure growth of S. pseudopneumoniae from sputum samples together with the great percentage of antibiotic resistance exhibited by those isolates observed in the present study should raise our attention to the clinical importance of this organism.The initial data recorded in this research concerning the prevalence, the genetic characterization, the antibiotic sensitivity pattern and the associated risk factors of S. pseudopneumoniae were the first in our locality as regarding this organism.Future studies should be encouraged to allow further description of S. pseudopneumoniae especially in relation to lower respiratory tract diseases; this may change the notion that they are just atypical pneumococci as it has been previously believed in our area.

Figure 1 .
Figure 1.Multiplex PCR results of the studied alpha hemolytic Streptococci including S. pseudopneumoniae isolates.Lane 1 shows the DNA standard marker.All lanes (2 to 9) show 522 bp fragments of positive internal control.Lane 10 is the negative control (distilled water).Lanes 2, 3 and 4 show the 290 bp bands from positive S. pseudopneumoniae isolates to aliB-like ORF2 gene.Lanes 5 and 6 show the 395 bp bands from positive S. pneumoniae isolates to lytA gene.Lanes 7, 8 and 9 show only the bands of positive internal control with absence of bands of other target genes from negative isolates of viridans group Streptococci.

Table 1 .
Primers used in the multiplex PCR of the studied isolates bp, Base pair.

Table 2 .
Multiplex PCR results of the examined alpha hemolytic Streptococcal isolates.

Table 3 .
Antibiotic sensitivity pattern of the studied 16 S. pseudopneumoniae isolates.

Table 4 .
distinctive features of patients with culture positive sputum for S. pseudopneumoniae.

Table 5 .
Patients risk factors for acquisition of S. pseudopneumoniae in relation to patients infected with other alpha hemolytic Streptococci.