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Pneumococcal empyema: Resistance patterns, fitness cost and serotype distribution

  • Amani Alnimr
    Correspondence
    Corresponding author. Amani Alnimr, MD, PhD, Department of Medical Microbiology, College of Medicine, King Fahad University Hospital, Imam Abdulrahman Bin Faisal University, P.O. Box 2114, Dammam 31451, Kingdom of Saudi Arabia.
    Affiliations
    Department of Microbiology, College of Medicine, King Fahad University Hospital, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
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      Abstract

      Background

      Streptococcus pneumoniae is a recognized etiology of invasive infections including parapneumonic empyema, and its resistance to antibiotics is evolving worldwide, raising concerns of encountering untreatable strains. This study measured the serotype distribution, antimicrobial susceptibility and biological cost incurred by resistance of pneumococci from pleural samples.

      Methods

      The serotype profiles, susceptibility results and growth rates were phenotypically determined for a panel of clinical strains of S. pneumoniae from cases of empyema between 2011 and 2019.

      Results

      Of 24 empyema cases, the isolated strains belonged to seven serotypes in the following descending order; 19A, 11A/D, 19F, 3, 7F, 1/6B while two strains remained non-typable. Penicillin susceptibility was shown in <80% of the isolates, while parenteral cephalosporins (cefuroxime and ceftriaxone) demonstrated activity in 83.3 and 95.8% respectively. High resistance frequency was noted for macrolides and sulfonamides, but the strains were uniformly sensitive to respiratory fluroquinolones, vancomycin and linezolid. The macrolide-resistant strain exhibited a high growth rate, suggesting a possible beneficial effect. Phenotypes with mono-resistance to sulfonamides and clindamycin were equally fit as the susceptible counterpart strains. Resistance to multiple antimicrobial agents resulted in a high degree of fitness deficit, while other resistant phenotypes were less fit.

      Conclusions

      The pneumococcal conjugate vaccine PCV13 serotypes still circulate in the community. The data indicate that resistance to certain antimicrobials incurs an apparent fitness cost in pneumococci which may limit the dissemination of such strains while low fitness cost, seen in case of resistance to macrolides, may contribute to the spread of resistant clones.

      Keywords

      Introduction

      Parapneumonic effusions and empyema are estimated to complicate around 2 to 3% of pneumonia cases, with a recent incremental trend.
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      ,
      • Nayak R.
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      • Lajkosz K.
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      Outcomes of Operative and Nonoperative Treatment of Thoracic Empyema: a Population-Based Study.
      Streptococcus pneumoniae is among the pyogenic bacteria commonly implicated in causing empyema worldwide, and particularly in community-acquired cases.
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      The microbiology of pleural infection in adults: a systematic review.
      Because of the difficulty to culture the fastidious organism, identification of pneumococcal serotypes that cause parapneumonic empyema is challenging.
      • Eltringham G.
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      • et al.
      Culture-negative childhood empyema is usually due to penicillin-sensitive Streptococcus pneumoniae capsular serotype 1.
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      It was proposed that serotype 1 possesses high invasive potential into the pleural space.
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      An epidemiological investigation of a sustained high rate of pediatric parapneumonic empyema: risk factors and microbiological associations.
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      Clinical features, aetiology and outcome of empyema in children in the north east of England.
      Further studies reported increases in the frequency of other serotypes (3, 7F, and 19A) amongst empyema cases in the post-vaccine era.
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      Distribution of Streptococcus pneumoniae serotypes that cause parapneumonic empyema in Turkey.
      The PCV7 was nationally introduced in 2009, and then replaced by PCV13 in 2011. Currently, the Saudi Thoracic Society pneumococcal vaccination guidelines recommend the use of PPSV23 for all vaccine-naïve healthy individuals, while high-risk adults aged ≥50 years should receive one dose of PCV13 followed by PPSV23 after ≥ 1 year. Children aged between 2 and 6 years with high-risk conditions also receive one dose of PPSV23 at least 8 weeks after the last PCV13.
      • Alharbi N.S.
      • Al-Barrak A.M.
      • Al-Moamary M.S.
      • et al.
      The Saudi Thoracic Society pneumococcal vaccination guidelines-2016.
      Introduction of vaccines has been linked not only to the reduction in the rate of the pneumococcal disease, but also the prevalence of drug-resistant pneumococcal strains.
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      • Farley M.M.
      • Hadler J.
      • et al.
      Active Bacterial Core Surveillance of the Emerging Infections Program Network. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine.
      The high genomic plasticity of S. pneumoniae makes its resistance to various classes of antibiotics a great problem.
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      • Farley M.M.
      • Hadler J.
      • et al.
      Active Bacterial Core Surveillance of the Emerging Infections Program Network. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine.
      Acquisition of antimicrobial resistance by the pathogen can be associated with a physiological fitness cost that results in a decreased transmission rate.
      • Gagneux S.
      Fitness cost of drug resistance in Mycobacterium tuberculosis.
      The fitness of a pathogen is a measure of the organism's ability to survive, reproduce, and be transmitted. Some drug resistance-conferring mutations have less impact on bacterial fitness. To survive and restore the ability to be transmitted, a mutant may revert to either susceptible or compensatory mechanisms accumulate over time, with the second being more likely.
      • Moore F.B.
      • Rozen D.E.
      • Lenski R.E.
      Pervasive compensatory adaptation in Escherichia coli.
      The fitness of a bacterium can be quantified in the laboratory by various in vitro models, including the average number of surviving progeny of a particular genotype in direct competition with a susceptible strain.
      • Pope C.F.
      • McHugh T.D.
      • Gillespie S.H.
      Methods to determine fitness in bacteria.
      Another approach to measure the fitness of resistant strains is to use the planktonic growth rate to quantify generation times against the wild type.
      • Pope C.F.
      • McHugh T.D.
      • Gillespie S.H.
      Methods to determine fitness in bacteria.
      Such methods, when used on clinical strains, can provide an epidemiological insight into the fitness status of commonly encountered strains.
      Data about fitness costs of resistant, invasive pneumococcal strains are very limited apart from few, inconclusive reports utilizing genetically transformed pneumococci rather than naturally occurring resistance in clinical isolates.
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      • Trzcinski K.
      • Thompson C.M.
      • Gilbey A.M.
      • et al.
      Incremental increase in fitness cost with increased beta -lactam resistance in pneumococci evaluated by competition in an infant rat nasal colonization model.
      Whereas the in vitro experiments contribute to the understanding of the pneumococcal population changes in response to various mutations, estimating fitness parameters in relation to antimicrobial resistance in clinical strains will be informative, since the biological cost of resistance mutations by various antimicrobial agents contributes to the selective pressure of an agent.
      • Pope C.F.
      • McHugh T.D.
      • Gillespie S.H.
      Methods to determine fitness in bacteria.
      In this study, pneumococcal serotypes that cause empyema in various age groups were evaluated along with their antimicrobial resistance and biological fitness patterns, to help improve the understanding of population dynamics of invasive pneumococcal isolates.

      Materials and methods

      Research settings and bacterial strains

      This is a prospective, observational study conducted at an academic medical institution in Alkhobar, Eastern Region of Saudi Arabia, during the nine-year period between January 1, 2011 and December 31, 2019. The Institutional Review Board approval was obtained (IRB 2014-01-012) ). All non-replicate, clinical strains of S. pneumoniae isolated from the pleural fluid or pleural biopsy of adults and children were included. Only cultures that were suggestive of true infections, rather than contamination, were considered based on clinical assessment, radiological findings and antimicrobial therapy. The samples were incubated on Columbia blood agar (CBA) (SPML, Saudi Arabia) in 5% CO2 at 35 °C for 18 h. The strains were identified to the species level based on colonial morphology with α-hemolysis, negative catalase test and susceptibility to a 6 mm optochin disk (5 micrograms) placed at the junction of the primary inoculum and second streak. The identity of S. pneumoniae strains was then confirmed by either VITEK 2 automated system (bioMe ́rieux Inc., Durham, NC, USA) based on biochemical signatures of the organisms or VITEK® MS (bioMe ́rieux Inc., Durham, NC, USA), an automated mass spectrometry microbial identification system that uses the matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) technology.

      Minimal inhibitory concentration determination

      E-test (AB-BIODISK, Sweden)-based minimal inhibitory concentration (MIC) was measured and interpreted following the manufacturer's instructions and the Clinical and Laboratory Standards Institute (CLSI 2019) guidance. Muller Hinton agar supplemented with 5% sheep blood (SPML, Saudi Arabia) was incubated overnight at 35 °C to determine the MIC using a 0.5 McFarland inoculum. The point of intersection of the inhibition ellipse on the E-test strip was recorded as the MIC. For the isolates where fitness studies were performed, the assays were performed in duplicate.

      Serotype distribution

      Serotyping of the isolates was carried out using the Pneumotest-Latex reaction based on a set of specific antisera in the Danish chessboard typing system (Statens Serum Institut, Copenhagen, Denmark) following manufacturer's recommendations. Briefly, an aliquot of 10 μL of a pneumococcal broth culture (BHI broth, SPML, Saudi Arabia) was mixed with 10 μL of latex reagent on a microscope slide, and manually rocked for 5–8 s. Positive reactions were read with the naked eye within 10 s, while agglutinations observed after more than 10 s were considered nonspecific and ignored.

      Fitness estimation by generation time

      The modified Youmans and Youmans method was used to measure the generation time in a planktonic growth rate model.
      • Pope C.F.
      • McHugh T.D.
      • Gillespie S.H.
      Methods to determine fitness in bacteria.
      Briefly, pneumococcal isolates and a control strain were separately incubated on CBA at 35 °C for 18 h in 5% CO2. Then, a single colony was inoculated in antibiotic-free BHI broth (5 mL), incubated for 4 h with frequent shaking to obtain exponential growth, and adjusted to turbidity comparable to a McFarland No. 0.5 standard. The culture was then diluted 1000-fold (approximately 105 CFU/ ml), loaded into the automated blood culture system BACTEC 9240 (BD Diagnostics, France) with a standard aerobic medium using 2 different dilutions, and incubated until they flagged positive. The time to positivity (TTP) was determined in minutes, followed by confirmation of purity by gram staining and spreading of one drop from the bottle contents onto CBA. The testing was done in triplicate, in two independent experiments. A modified Youmans and Youmans method was applied to calculate the growth rate constant (K) and generation time (G), using the formula K = (logA - logB)/t, where A is the higher dilution, B is the lower dilution and t is equal to the difference in the mean of TTP for three replicates. The generation time was then calculated using the equation: G = log 2/K.
      • Pope C.F.
      • McHugh T.D.
      • Gillespie S.H.
      Methods to determine fitness in bacteria.

      Statistical analysis

      Differences in fitness between the various phenotypes and control strains were analyzed by the one-way analysis of variance (ANOVA) test using the GraphPad Prism version 9.3.1 for Mac, which compares the three means. The null hypothesis was all population means were equal, and the alternative hypothesis was that at least one mean was different. A p value of less than 0.05 was considered significant in the calculated generation times of different strains.

      Results

      In total, 24 isolates of S. pneumoniae were collected from invasive pleural samples exhibiting various susceptibility patterns (Table 1). All the pneumococcal isolates were susceptible to respiratory fluroquinolones, linezolid and the tested glycopeptide, vancomycin. They demonstrated variable resistance levels to other classes of antibiotics in the following ascending order: cefotaxime (a 3rd generation cephalosporin), parenteral cefuroxime, oral cefuroxime/parenteral penicillin, oral penicillin, doxycycline, clindamycin (lincosamide), and trimethoprim/sulfamethoxazole, with the highest resistance rate seen with macrolides (erythromycin) in 70.4% of the strains.
      TABLE 1The proportion of resistance among 24 respiratory strains of Streptococcus pneumoniae isolated from adults.
      DrugSusceptible number (%)
      Penicillin: parenteral dose

      oral dose
      19 (79.2%)

      18 (75%)
      Cefuroxime: parenteral dose

      oral dose
      20 (83.3%)

      19 (79.2%)
      Cefotaxime23 (95.8%)
      Erythromycin7 (29.6%)
      Clindamycin15 (62.5%)
      Levofloxacin24 (100%)
      Moxifloxacin24 (100%)
      Doxycycline17 (70.8%)
      Trimethoprim-sulfamethoxazole8 (33.3%)
      Vancomycin24 (100%)
      Linezolid24 (100%)
      The serotypes detected were as follows: six 19A strains (25%), five 11A/D strains (20.8%), four 19F strains (16.7%), three serotype 3 (12.5%), two 7F strains (8.3%), one serotype 1 (4.2%), and one serotype 6B (4.2%) while two strains remained phenotypically non-typable (8.3%). The characteristics of the cases from whom the strains were obtained are illustrated in Table 2.
      TABLE 2Clinical, epidemiological and laboratory characteristics of the 24 empyema cases.
      VariableFindings
      Median age (years) ± SD59.5 ± 14.3
      < 18 years10 (41.7%)
      ≥ 18 years14 (58.3%)
      Gender
      Male15 (62.5%)
      Female9 (37.5%)
      Blood culture
      Performed22 (91.7%)
      Positive1 (4.2%)
      Sputum culture
      Performed24 (100%)
      Positive2 (8.3%)
      Comorbidities
      Hypertension4 (16.7%)
      Diabetes mellitus5 (20.8%)
      Chronic cardiac condition1 (4.2%)
      Autoimmune diseases1 (4.2%)
      Malignancy1 (4.2%)
      Sickle cell disease2 (8.3%)
      Sepsis3 (12.5%)
      ICU admission5 (20.8%)
      LOS (days)19.3 ± 13.6
      In-hospital mortality2 (8.3%)
      90-day mortality3 (12.5%)
      Abbreviations: ICU, intensive care unit; LOS, length of stay.
      Isolates exhibiting monoresistance to each class of antimicrobials were further characterized by measuring their MICs as shown in Table 3, as well as an isolate exhibiting multidrug resistance (penicillin, erythromycin and trimethoprim-sulfamethoxazole).
      TABLE 3Quantitative antibiotic susceptibilities (µg/ml) of Streptococcus pneumoniae ATCC 49,619 and a panel of clinical strains (SN): * denotes resistance. MIC represents the minimal inhibitory concentration of antimicrobial agents tested against the organism.
      StrainATCC 49,619SN-24SN-18SN-109SN-45SN-30SN-7SN-149SN-12
      Serotype19F11A/D19A6B119FNT*319A
      Resistance PhenotypePansusceptiblePansusceptiblePenicillin- resistantCefotaxime resistantMacrolide-resistantMacrolide-lincosamide resistantDoxycycline-resistantSulfonamide monoresistantMDR strain
      Penicillin0.030.063*11.50.50.250.516*
      Cefotaxime0.250.1250.2560.250.250.250.1250.5
      Erythromycin0.060.060.1250.25256*4*0.1250.2548*
      Clindamycin0.030.060.060.250.1253*0.1250.2516*
      Levofloxacin0.50.25120.51.50.5316*
      Moxifloxacin0.060.1250.2510.1250.750.2514
      Doxycycline0.060.060.1250.250.1250.2516*0.250.25
      Trimethoprim-sulfamethoxazole0.5/9.50.5/9.51/92/381/92/381/94/76*4/76*
      Vancomycin0.120.0030.0030.50.50.0030.0060.250.5
      Table 4 demonstrates the variable effects of antibiotic resistance patterns in pneumococci on their mean generation times measured in minutes. The strains were collected from unrelated patients and therefore represented nonclonal isolates as their epidemiological picture and serotypes suggested.
      TABLE 4Association between antibiotic resistance patterns of pneumococci and their mean generation times measured in minutes.
      PhenotypeGeneration time in min +/- SEMP value
      ATCC 4961928.7 (26.5 - 30.8)
      Pansusceptible31 (28.9 - 31.3)0.33
      Penicillin resistant strain40.4 (32.8 - 47.3)0.45
      Cefotaxime-resistant strain48.3 (42.9 - 55.4)0.01
      Macrolide-resistant strain18.4 (17.5 – 19.4)0.02
      Macrolide-lincosamide resistant strain28.2 (25.7 – 30.9)0.39
      Doxycycline-resistant strain52.1 (49.9 – 53.7)0.0002
      Sulfonamide monoresistant strain29.0 (26.3 - 32.9)0.10
      MDR strain73.2 (65.2 - 77.6)0.035

      Discussion

      S. pneumoniae remains a leading cause of a wide range of invasive infections including empyema, even after the introduction of pneumococcal vaccines.
      • Whitney C.G.
      • Farley M.M.
      • Hadler J.
      • et al.
      Active Bacterial Core Surveillance of the Emerging Infections Program Network. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine.
      Identifying invasive pneumococcal strains is a prerequisite for efficient implementation of regional and global immunization programs. Therefore, this study assessed the serotype distribution of pneumococci implicated in causing 24 empyema cases in a leading hospital in Eastern Saudi Arabia between 2011 and 2019. In an older multicenter study conducted in Saudi Arabia between 2000 and 2004 that included 350 pediatric pneumococcal isolates from various invasive sites, the most common serotype was 14, followed by serotypes 23F, 6B, and 19F accounting for > 80% of the invasive disease.
      • Shibl A.M.
      Distribution of serotypes and antibiotic resistance of invasive pneumococcal disease isolates among children aged 5 years and under in Saudi Arabia (2000-2004).
      The common serotypes detected in order of frequency in the current study were 19A, 11A/D, 19F, 3, 7F, and 1/6B Notably, four of these six serotypes are included in the PCV13. It was reported earlier that serotype 3, a PCV13 vaccine strain, still circulates worldwide.
      • Antachopoulos C.
      • Tsolia M.N.
      • Tzanakaki G.
      • et al.
      Parapneumonic pleural effusions caused by Streptococcus pneumoniae serotype 3 in children immunized with 13-valent conjugated pneumococcal vaccine.
      ,
      • Olarte L.
      • Barson W.J.
      • Barson R.M.
      • et al.
      Pneumococcal Pneumonia Requiring Hospitalization in US Children in the 13-Valent Pneumococcal Conjugate Vaccine Era.
      The non-PCV7 serotype 19A was the most prevalent in our cohort, and it has been also described as a cause of invasive pneumococcal disease following the use of PCV7.
      • Pelton S.I.
      • Huot H.
      • Finkelstein J.A.
      • et al.
      Emergence of 19A as virulent and multidrug resistant Pneumococcus in Massachusetts following universal immunization of infants with pneumococcal conjugate vaccine.
      Serotype 7F has been associated with increased morbidity and mortality seen with pneumococcal infections.
      • Rückinger S.
      • von Kries R.
      • Siedler A.
      • et al.
      Association of serotype of Streptococcus pneumoniae with risk of severe and fatal outcome.
      Another focus of this work was to examine the susceptibility patterns of S. pneumoniae isolated from pleural samples. Early studies conducted in Saudi Arabia have shown high prevalence of drug-resistant S. pneumoniae isolates.
      • Shibl A.M.
      Distribution of serotypes and antibiotic resistance of invasive pneumococcal disease isolates among children aged 5 years and under in Saudi Arabia (2000-2004).
      ,
      • Twum-Danso K.
      • Al-Mazrou A.M.
      • Kambal A.M.
      • et al.
      Penicillin resistance in serogroups/serotypes of Streptococcus pneumoniae causing invasive infections in Central Saudi Arabia.
      ,
      • Memish Z.A.
      • Balkhy H.H.
      • Shibl A.M.
      • et al.
      Streptococcus pneumoniae in Saudi Arabia: antibiotic resistance and serotypes of recent clinical isolates.
      Table 1 shows that the resistance of pneumococci to B-lactams is less than the findings of previous reports. This can be explained by both the revision of the interpretative breakpoints of B-lactam resistance in non-meningeal pneumococci by CLSI in 2008, in addition to the possible impact of fitness cost on the survival of the resistant strains in the community (Table 3). The CLSI revisions resulted in the different categorizations based on the new guidelines examining the efficacy of double-dose penicillin on respiratory strains with intermediate MICs based on the older breakpoints.
      • Mandell L.A.
      • Wunderink R.G.
      • Anzueto A.
      • et al.
      Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults.
      Our resistance rates are higher than those described in the United States using current breakpoints, where at present 3.1% and 2.1% of non-meningeal pneumococcal strains are intermediate and resistant to penicillin respectively.

      Active Bacterial Core Surveillance (ABCs): emerging Infections Program Network. Streptococcus pneumoniae, 2014. http://www.cdc.gov/abcs/reports-findings/survreports/spneu14.html (Accessed on April 20, 2022).

      On the other hand, higher rates of penicillin resistance have been reported in Asian countries. A multi-center review of pneumococcal isolates (n = 685) from 14 centers in 11 Asian countries over eighteen months (January 2000 to June 2001) illustrated an overall 52% non-susceptibility to penicillin, with a rate of resistance as high as 74% in Vietnam.
      • Song J.H.
      • Jung S.I.
      • Ko K.S.
      • et al.
      High prevalence of antimicrobial resistance among clinical Streptococcus pneumoniae isolates in Asia (an ANSORP study).
      In comparison, the resistance rates to both macrolide and sulfonamide groups of antibiotics (Table 1) are still alarming (70.3% and 66.6 respectively). The minimal fitness cost of drug resistance in these strains (GT = 18.4 and 29.0 min respectively) was also noted (Table 4). Pneumococcal resistance to sulfonamides is encountered in one third of the isolates in the United States, with emergence of drug-resistant replacement strains following the widespread use of the pneumococcal conjugate vaccine. The most prevalent replacement strain (serotype 19A) showed a high frequency of resistance to sulfonamides (83%) in comparison to other serotypes, 35B and 7F, that remained uniformly susceptible.
      • Jacobs M.R.
      • Good C.E.
      • Windau A.R.
      • et al.
      Activity of ceftaroline against recent emerging serotypes of Streptococcus pneumoniae in the United States.
      For macrolides, considerable geographical variation in pneumococcal resistance rates is noted.

      Active Bacterial Core Surveillance (ABCs): emerging Infections Program Network. Streptococcus pneumoniae, 2014. http://www.cdc.gov/abcs/reports-findings/survreports/spneu14.html (Accessed on April 20, 2022).

      ,
      • Kim L.
      • McGee L.
      • Tomczyk S.
      • et al.
      Biological and Epidemiological Features of Antibiotic-Resistant Streptococcus pneumoniae in Pre- and Post-Conjugate Vaccine Eras: a United States Perspective.
      Introduction of the conjugate vaccine didn't decrease the resistance patterns since the emergent replacement strains have a high rate of macrolide resistance.
      • Simões A.S.
      • Pereira L.
      • Nunes S.
      • et al.
      Clonal evolution leading to maintenance of antibiotic resistance rates among colonizing Pneumococci in the PCV7 era in Portugal.
      Widespread use of fluoroquinolones increases the risk for emergence of resistance.
      • Kupronis B.A.
      • Richards C.L.
      • Whitney C.G.
      Active Bacterial Core Surveillance Team. Invasive pneumococcal disease in older adults residing in long-term care facilities and in the community.
      High rates of fluroquinolones resistance have been reported in Belgium, Croatia, and Hong Kong where 3–13% of pneumococci were reported resistant.
      • Pankuch G.A.
      • Bozdogan B.
      • Nagai K.
      • et al.
      Incidence, epidemiology, and characteristics of quinolone-nonsusceptible Streptococcus pneumoniae in Croatia.
      • Ho P.L.
      • Yung R.W.
      • Tsang D.N.
      • et al.
      Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in 2000.
      • Ceyssens P.J.
      • Van Bambeke F.
      • Mattheus W.
      • et al.
      Belgian Streptococcus pneumoniae Study Group, Tulkens PM, Vanhoof R. Molecular Analysis of Rising Fluoroquinolone Resistance in Belgian Non-Invasive Streptococcus pneumoniae Isolates (1995-2014).
      In contrast, no resistance has been reported in Germany.
      • Pletz M.W.
      • van der Linden M.
      • von Baum H.
      • et al.
      CAPNETZ study group. Low prevalence of fluoroquinolone resistant strains and resistance precursor strains in Streptococcus pneumoniae from patients with community-acquired pneumonia despite high fluoroquinolone usage.
      There was no resistance to fluroquinolones, linezolid or vancomycin detected in our cohort, although vancomycin tolerance has been described for some strains in a previous study leading to therapeutic failure.
      • Olivaris A.
      • Trejo J.
      • Arellano-Galindo J.
      • et al.
      pep27 and lytA in Vancomycin-Tolerant Pneumococci.
      A limitation of the study is the small number of cases due to the low culture yield of S. penumoniae and thus culture-negative pneumococcal empyema could have been missed, driving an effect on the serotype epidemiology. Additionally, the vaccination status could not be retrieved for those cases. Despite the high vaccination rates in Saudi Arabia, the patients may have been older than the requisite age for routine vaccination when the PCV7 and PCV13 were introduced. Furthermore, pre-vaccination serotypes for empyema cases in the region are not known. Nevertheless, the data provided insights into the serotype distribution of empyema cases across a period of nine years.

      Conclusions

      In conclusion, the study has shed light on the overall resistance patterns and serotype distribution of pneumococcal empyema, and the fitness of resistant strains in comparison to the sensitive counterparts. The contribution of transmission to the spread of drug resistance is likely in cases of resistance to the classes of antimicrobials with minimal impact on organism's fitness. To the best of our knowledge, this is the first study where generation times of clinical pneumococcal strains are measured to provide evidence of biological burden of antimicrobial resistance. Surveillance of invasive pneumococcal strains is worth continuing to guide future vaccine development, and further work is suggested to assess the genotype-based fitness in circulating strains within the community.

      Declaration of Competing Interest

      The author declares no conflicts of interest.

      Acknowledgments

      This work was supported by grant 2013216 from the Deanship of Scientific Research at Imam Abdulrahman Bin Faisal University. Sincere thanks to the technical assistance provided by Ms. Rosalinda Sastrillo.

      Author Contributions

      A.A. conceived the idea, collected the data, performed the analysis, and wrote the paper.

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