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Pulmonary Disease Associated With Nonencapsulated Streptococcus pneumoniae

Pulmonary Disease Associated With Nonencapsulated Streptococcus pneumoniae Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Open Forum Infectious Diseases DOI: 10.1093/ofid/ofy135 BRIEF REPORT Pulmonary Disease Associated With Nonencapsulated Streptococcus pneumoniae Caleb S. Martin, Jessica L. Bradshaw, Haley R. Pipkins, and Larry S. McDaniel Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, Mississippi We discuss 3 patients presenting with pneumonia associated with nonencapsulated Streptococcus pneumoniae (NESp), an emerging pathogen commonly causing upper respiratory infections. Clinical isolates obtained from these patients were characterized to evalu- ate their respective antibiotic resistance and virulence mechanisms. We demonstrate that NESp resistant to classical drug treatments are isolated during pneumonia. Keywords. AliD; NESp, pneumococcus, pneumonia, PspK. Streptococcus pneumoniae (pneumococcus) is a bacterium that Surveillance Lab (Jackson, MS) between October 2015 and July asymptomatically colonizes the human nasopharynx but also con- 2016. S. pneumoniae identification was verified by alpha hemoly- sistently causes diseases such as otitis media and pneumonia [1, sis and optochin sensitivity. The previously characterized UMMC 2]. Currently licensed vaccines protect against pneumococcal dis- NESp clinical isolate C144.66 was included in this study for com- ease associated with encapsulated strains but elicit no protection parison purposes [10]. Antimicrobial susceptibility testing of against nonencapsulated S. pneumoniae (NESp) [3]. Consequently, bacterial cultures using VITEK 2 was completed by the UMMC increased vaccine use has selectively increased isolation of NESp microbiology clinical lab. Patient characteristics, sources of infec- that encode the virulence-associated genes pspK, aliC, and aliD tion, and relative antibiotic resistance are listed in Table 1. [4, 5]. Understanding how NESp establish infections is essential to Polymerase Chain Reaction preventing NESp-associated disease. Additionally, analyzing links Polymerase chain reaction (PCR) amplification was performed between patient comorbidities and susceptibility to pneumococcal with primers for the conserved capsule gene cpsA (forward: 5’- infection could improve prophylactic patient care. Mississippi has GCAGTACAGCAGTTTGTTGGACTGACC-3’ and reverse: the highest vaccination rates in the United States, at >97.4% cover- 5’-GAATATTTTCATTATCAGTCCCAGTC-3’) or nonencapsu- age [6]. Mississippi also has a high prevalence of chronic diseases, lated genes pspK (for ward: GCAAA TCA GCCA GT AA CTGTGA -3’ including chronic obstructive pulmonary disease (COPD), hyper- and reverse: 5’-CAAGATAAGCTTTCTGCACCTCT-3’), aliC tension, diabetes, and obesity [7–9]. Due to vaccine-induced selec- (forward: 5’-GACCAGATTACCAAGATCCAGCAAC-3’ and tive pressure and high disease rates in Mississippi, the aim of this reverse: 5’-GCCCTTTGTTATACCTAGATGTTTC-3’), and study was to determine NESp prevalence among Mississippians aliD (forward: 5’-AGATGCCAAATGGTTCACGGCA-3’ and presenting with pneumococcal disease and determine the viru- reverse: 5’GGTCGTCAATGGCCTTCACC-3’). The encapsu- lence mechanisms associated with isolated NESp. lated pneumococcal strain WU2 was used as a positive control + + for cpsA amplification. NESp strains MNZ41 (aliC aliD ) and METHODS MNZ67 (pspK ) were used as positive controls for amplifica- Clinical Isolates tion of aliC, aliD, and pspK. PCR products were amplified using Thirty-five S. pneumoniae clinical isolates were retrieved from the GoTaq (Promega) with the cycling parameters suggested by the University of Mississippi Medical Center (UMMC) Microbiology manufacturer and an annealing temperature of 52°C. Amplified PCR products were analyzed on a 1% agarose gel with ethidium Received 25 May 2018; editorial decision 3 June 2018; accepted 5 June 2018. bromide staining. Correspondence: L.  S. McDaniel, PhD,  Department of Microbiology and Immunology, University of Mississippi Medical Center,  2500 N State Street, Jackson, MS 39216 (lmcdan- iel@umc.edu). Biofilm Formation Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases A 24-well plate was seeded with 10 colony forming units (CFU) Society of America. pneumococci in biofilm media (Todd-Hewitt broth with 0.5% This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which yeast extract, 8 U/mL catalase, and 10% horse serum) per well and permits non-commercial reproduction and distribution of the work, in any medium, provided the incubated at 37°C with 5% CO for 24 hours. Following incuba- original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com tion, biofilm media was removed from the wells, and biofilms were BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Table 1. Clinical Correlation and Characteristics of NESp Isolated From Patients CA vs Virulence CI Source Age/Sex Presentation/Comorbidity Immunization HA Antibiotic Resistance Gene Encoded C144.66 Middle ear 2/M Chronic adenoiditis ND CA Erythromycin pspK Penicillin Sulfamethoxazole/trimethoprim 361.67 Pulmonary 48/M Intubation PPSV HA Clindamycin pspK ARF 2015 Tetracycline Erythromycin 327.251 Pulmonary 66/M COPD PPSV CA Penicillin aliD Diabetes 2013 and 2016 264.174 Pulmonary 5/F Cystic fibrosis Not immunized CA Erythromycin aliD Abbreviations: ARF, acute respiratory failure; CA, community-acquired; CI, clinical isolate; COPD, chronic obstructive pulmonary disease; HA, hospital-acquired; ND, not determined; NESp, nonencapsulated Streptococcus pneumoniae; PPSV, pneumococcal polysaccharide vaccine (Pneumovax23). stained with 350 µL of 0.1% crystal violet at room temperature for pspK whereas 327.251 and 264.174 encoded aliD (Table 1). All 3 30 minutes. Unbound crystal violet was carefully removed, 1 mL newly identified NESp isolates were associated with pulmonary of 100% ethanol was added to each well to solubilize biofilm-as- disease (Table 1); 361.67 was associated with acute pneumonia sociated crystal violet, and the plate was shaken at room tempera- in an adult male following intubation during acute respiratory ture for 10 minutes. Solubilized crystal violet was transferred to a failure, and 327.251 was isolated during a COPD exacerbation 96-well plate that was measured at OD with an xMark microplate in a 66-year-old male. Both adult male patients were previ- spectrophotometer (BioRad). All samples were assayed in tripli- ously vaccinated against pneumococcus (Table 1); 264.174 was cate, and independent experiments were performed twice. isolated from a cystic fibrosis (CF) pediatric patient who had received no prior vaccination against pneumococcus. The “gold Pneumococcal Adherence and Invasion of Epithelial Cells standard” treatment for S.  pneumoniae infections is penicillin, Human Detroit 562 pharyngeal and A549 pulmonary epithe- and 2 out of the 4 strains were found to be resistant to penicillin lial cells were grown to approximately 90% confluence in 10% (Table  1). NESp strains were also resistant to antibiotics com- fetal calf serum (FCS)–supplemented Eagle’s minimal essential monly used to treat S. pneumoniae infections, such as erythro- medium (EMEM) with 100 µg/mL penicillin, 50 µg/mL strepto- mycin and sulfamethoxazole/trimethoprim. mycin, and 100 µg/mL amphotericin B in 24-well plates at 37°C NESp Phenotypes Permit Persistence in a Host with 5% CO . Pneumococcal adherence and invasion assays PspK is an adhesin that aids in adherence to host cells, and AliC were performed as previously described [11]. Briefly, 10 pneu- and AliD are oligopeptide binding proteins that sense the extra- mococci suspended in antibiotic-free EMEM was added to each cellular environment [5]. We grouped isolates based on expres- well. Adhered pneumococci were enumerated after 30 minutes sion of PspK or AliD to examine virulence mechanisms. Human of incubation with human epithelial cells. To assess invasion, pharyngeal and pulmonary epithelial cells were used to mimic pneumococci were incubated with epithelial cells for 2 hours, the host niche during colonization and pneumonia (Figure 1). All followed by elimination of extracellular bacteria with 200 µg/mL clinical isolates efficiently adhered to pharyngeal cells (Figure 1A). gentamycin before lysing epithelial cells and enumerating intra- PspK-expressing 361.67 adhered to A549 pulmonary cells signif- cellular pneumococcal CFUs. Two independent experiments icantly more (P < .001) than all other isolates (Figure 1B). PspK- were performed, and all samples were assayed in triplicate. expressing cells invaded both pharyngeal and pulmonary cells Statistical Analysis significantly more (P < .001) than their AliD-expressing counter- PRISM 5 software (GraphPad Software, Inc.) was used to analyze parts (Figure 1C and D). When comparing epithelial cell invasion the results. To assess if there were any significant differences in by PspK-expressing strains C144.66 and 361.67, C144.66 demon- the means between all groups compared, we used one-way ana- strated significantly greater (P < .01) invasion of pharyngeal cells lysis of variance (ANOVA). Tukey post-tests allowed us to iden- (Figure 1C), whereas 361.67 demonstrated significantly greater (P tify the specific significant differences in the groups analyzed. < .05) invasion of pulmonary cells (Figure 1D). All NESp strains A P value of less than .05 was considered statistically significant. formed dense biofilms (Figure 1D), but the AliD-expressing strain 327.251 formed significantly more biofilm than the other pulmo- RESULTS nary isolates 361.67 (P < .05) and 264.174 (P < .01). Survey of Pneumococcal Isolates and Clinical Correlation DISCUSSION PCR analysis of the capsule locus demonstrated that 3/35 (~9%) pneumococcal isolates lacked the highly conserved capsule In this report, we determined that nearly 9% of pneumococcal gene cpsA. Further PCR analysis revealed that 361.67 encoded infections at UMMC were linked to NESp, and we described 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 AB Detroit 562 adhesion A549 adhesion 5.5 5.5 *** 5.0 5.0 4.5 4.5 4.0 4.0 3.5 3.5 C144.66 361.67 327.251 264.174 C144.66 361.67 327.251 264.174 pspK aliD pspK aliD C Detroit 562 invasion D A549 invasion *** 3.0 3.0 ** *** *** 2.5 2.5 *** 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 C144.66 361.67 327.251 264.174 C144.66 361.67 327.251 264.174 pspK aliD pspK aliD NESp biofilm formation 0.18 ** 0.16 0.14 0.12 0.10 0.08 0.06 C144.66 361.67 327.251 264.174 pspK aliD Figure 1. Nonencapsulated Streptococcus pneumoniae (NESp) phenotypes influencing pulmonary disease. Human Detroit 562 pharyngeal (A and C) and A549 pulmonary (B and D) epithelial cells were used to assess NESp adherence (A and B) and invasion (C and D). Biofilms formed (E) after 24-hour growth in 24-well plates were assayed using crystal violet staining and measuring the optical density at 630 nm. A, All clinical isolates efficiently adhered to pharyngeal cells. B, Clinical isolate (CI) 361.67 adhered to pulmonary cells significantly more than all other isolates. PspK-expressing cells invaded both pharyngeal (C) and pulmonary (D) cells at low rates. CI C144.66 invaded pharyngeal cells more efficiently than 361.67 (C), whereas 361.67 invaded pulmonary cells more efficiently than C144.66 (D). CI 327.251 formed significantly more biofilm than * ** *** 361.67 or 264.174. Data represent 2 independent studies performed in triplicate. Error bars denote standard error of the mean ( P < .05; P < .01; P < .001). Abbreviations: CFU, colony forming units; OD, optical density. 3 patients who develop NESp-associated pneumonia. We also how NESp are able to establish disease, we examined virulence demonstrated that NESp express antibiotic-resistant phenotypes mechanisms of the clinical isolates. All NESp isolates efficiently that threaten classical treatment measures. As there are no cur- adhered to pharyngeal cells and formed dense biofilms, which rently licensed NESp preventatives, antibiotic-resistant NESp likely aided in nasopharyngeal colonization and development are an emerging threat to public health. To better understand of pneumonia. NESp did not invade epithelial cells efficiently, NESp Associated Bacterial Pneumonia • OFID • 3 OD 630 nm Log CFU/mL Log CFU/mL Log CFU/mL Log CFU/mL Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 which suggests that NESp are able to withstand the extracellu- risk of diseases associated with these strains. Further surveil- lar environment despite the lack of a protective polysaccharide lance of NESp isolates will need to be conducted to determine capsule. the widespread NESp threat to public health and NESp-specific In the patient infected with 361.67, translocation of 361.67 virulence mechanisms. to the lungs during intubation could have led to the infection, which is a phenomenon that has been previously reported [12]. Acknowledgments As PspK has been shown to increase adherence to pulmonary Financial support. This work was supported by the Medical Scholars Research Program at the University of Mississippi Medical Center and insti- epithelial cells and persistence in a murine lung model, PspK of tutional research funds. 361.67 may have also facilitated persistence during pulmonary Coni fl cts of interest. None declared. infection [11]; 361.67 had greater adherence to A549 pulmo- nary cells when compared with a clinical isolate also expressing References PspK (C144.66), which may have been due to diverse genetic 1. Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneu- backgrounds or differential expression patterns that increase moniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008; 6:288–301. interactions with epithelial cells. C144.66 also invaded phar- 2. Musher DM. Infections caused by Streptococcus pneumoniae: clinical spectrum, yngeal cells significantly more than 361.67 and caused chronic pathogenesis, immunity, and treatment. Clin Infect Dis 1992; 14:801–7. 3. Castiglia P.  Recommendations for pneumococcal immunization outside routine adenoiditis rather than pneumonia. Even though both these childhood immunization programs in Western Europe. Adv Ther 2014; 31:1011–44. strains express PspK, NESp may have a predilection for cer- 4. Croucher NJ, Harris SR, Fraser C, et  al. Rapid pneumococcal evolution in response to clinical interventions. Science 2011; 331:430–4. tain anatomic and physiologic environments. NESp strains iso- 5. Keller LE, Robinson DA, McDaniel LS. Nonencapsulated Streptococcus pneumo- lated during conjunctivitis encode niche-specific adaptations, niae: emergence and pathogenesis. mBio 2016; 7:1–12. 6. Seither R, Calhoun K, Knighton CL, et al. Vaccination coverage among children and similar adaptations likely occur during middle ear or lung in kindergarten - United States, 2014–15 school year. MMWR Morb Mortal Wkly infections [13]. NESp strains expressing AliD showed no inva- Rep 2015; 64:897–904. sive potential in vitro but caused pulmonary infections in the 7. Dwyer-Lindgren L, Bertozzi-Villa A, Stubbs RW, et al. Trends and patterns of dif- ferences in chronic respiratory disease mortality among US counties, 1980–2014. clinical setting. The altered lung environment in COPD and JAMA 2017; 318:1136. cystic fibrosis patients may be potentiating these NESp infec- 8. Fang J, Gillespie C, Ayala C, Loustalot F. Prevalence of self-reported hypertension and antihypertensive medication use among adults aged ≥18 years -United States, tions by supplying an altered host niche that supports NESp 2011–2015. MMWR Morb Mortal Wkly Rep 2018; 67:219–24. growth; for instance, chronic hypersecretion of mucus and air 9. Mokdad AH, Bowman BA, Ford ES, et  al. The continuing epidemics of obesity and diabetes in the United States. JAMA 2001; 286:1195–200. trapping that prevents proper expansion of the lungs predispose 10. Dixit C, Keller LE, Bradshaw JL, et al. Nonencapsulated Streptococcus pneumoniae this site to infection [14]. Also, invasive pneumococcal disease as a cause of chronic adenoiditis. IDCases 2016; 4:56–8. 11. Pipkins HR, Bradshaw JL, Keller LE, McDaniel LS. Increased virulence of an caused by encapsulated strains has been shown to be more prev- encapsulated Streptococcus pneumoniae upon expression of pneumococcal sur- alent in patients with a chronic pulmonary condition, and this face protein K. J Infect Dis 2018; 217:1637–44. is likely the case for NESp as well [15]. Pneumococcal vaccina- 12. Gardner JG, Rueda AM, Musher DM. Acute onset of pneumococcal pneumonia following instrumentation of the respiratory tract. Open Forum Infect Dis 2018; tions are currently recommended for patients who are immu- 5:ofy047. nosuppressed or diagnosed with chronic pulmonary diseases. 13. Valentino MD, McGuire AM, Rosch JW, et  al. Unencapsulated Streptococcus pneumoniae from conjunctivitis encode variant traits and belong to a distinct Vaccinating these individuals reduces the risk of pneumococcal phylogenetic cluster. Nat Commun 2014; 5:5411. disease associated with vaccine-covered serotypes but increases 14. Lange P. Chronic obstructive pulmonary disease and risk of infection. Pneumonol Alergol Pol 2009; 77:284–8. the risk of NESp-associated disease in these patients. Overall, 15. Inghammar M, Engström G, Kahlmeter G, et al. Invasive pneumococcal disease NESp strains are becoming more prominent human pathogens, in patients with an underlying pulmonary disorder. Clin Microbiol Infect 2013; 19:1148–54. and patients with certain comorbid conditions are at a higher 4 • OFID • BRIEF REPORT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

Pulmonary Disease Associated With Nonencapsulated Streptococcus pneumoniae

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Society of America.
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2328-8957
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10.1093/ofid/ofy135
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Abstract

Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Open Forum Infectious Diseases DOI: 10.1093/ofid/ofy135 BRIEF REPORT Pulmonary Disease Associated With Nonencapsulated Streptococcus pneumoniae Caleb S. Martin, Jessica L. Bradshaw, Haley R. Pipkins, and Larry S. McDaniel Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, Mississippi We discuss 3 patients presenting with pneumonia associated with nonencapsulated Streptococcus pneumoniae (NESp), an emerging pathogen commonly causing upper respiratory infections. Clinical isolates obtained from these patients were characterized to evalu- ate their respective antibiotic resistance and virulence mechanisms. We demonstrate that NESp resistant to classical drug treatments are isolated during pneumonia. Keywords. AliD; NESp, pneumococcus, pneumonia, PspK. Streptococcus pneumoniae (pneumococcus) is a bacterium that Surveillance Lab (Jackson, MS) between October 2015 and July asymptomatically colonizes the human nasopharynx but also con- 2016. S. pneumoniae identification was verified by alpha hemoly- sistently causes diseases such as otitis media and pneumonia [1, sis and optochin sensitivity. The previously characterized UMMC 2]. Currently licensed vaccines protect against pneumococcal dis- NESp clinical isolate C144.66 was included in this study for com- ease associated with encapsulated strains but elicit no protection parison purposes [10]. Antimicrobial susceptibility testing of against nonencapsulated S. pneumoniae (NESp) [3]. Consequently, bacterial cultures using VITEK 2 was completed by the UMMC increased vaccine use has selectively increased isolation of NESp microbiology clinical lab. Patient characteristics, sources of infec- that encode the virulence-associated genes pspK, aliC, and aliD tion, and relative antibiotic resistance are listed in Table 1. [4, 5]. Understanding how NESp establish infections is essential to Polymerase Chain Reaction preventing NESp-associated disease. Additionally, analyzing links Polymerase chain reaction (PCR) amplification was performed between patient comorbidities and susceptibility to pneumococcal with primers for the conserved capsule gene cpsA (forward: 5’- infection could improve prophylactic patient care. Mississippi has GCAGTACAGCAGTTTGTTGGACTGACC-3’ and reverse: the highest vaccination rates in the United States, at >97.4% cover- 5’-GAATATTTTCATTATCAGTCCCAGTC-3’) or nonencapsu- age [6]. Mississippi also has a high prevalence of chronic diseases, lated genes pspK (for ward: GCAAA TCA GCCA GT AA CTGTGA -3’ including chronic obstructive pulmonary disease (COPD), hyper- and reverse: 5’-CAAGATAAGCTTTCTGCACCTCT-3’), aliC tension, diabetes, and obesity [7–9]. Due to vaccine-induced selec- (forward: 5’-GACCAGATTACCAAGATCCAGCAAC-3’ and tive pressure and high disease rates in Mississippi, the aim of this reverse: 5’-GCCCTTTGTTATACCTAGATGTTTC-3’), and study was to determine NESp prevalence among Mississippians aliD (forward: 5’-AGATGCCAAATGGTTCACGGCA-3’ and presenting with pneumococcal disease and determine the viru- reverse: 5’GGTCGTCAATGGCCTTCACC-3’). The encapsu- lence mechanisms associated with isolated NESp. lated pneumococcal strain WU2 was used as a positive control + + for cpsA amplification. NESp strains MNZ41 (aliC aliD ) and METHODS MNZ67 (pspK ) were used as positive controls for amplifica- Clinical Isolates tion of aliC, aliD, and pspK. PCR products were amplified using Thirty-five S. pneumoniae clinical isolates were retrieved from the GoTaq (Promega) with the cycling parameters suggested by the University of Mississippi Medical Center (UMMC) Microbiology manufacturer and an annealing temperature of 52°C. Amplified PCR products were analyzed on a 1% agarose gel with ethidium Received 25 May 2018; editorial decision 3 June 2018; accepted 5 June 2018. bromide staining. Correspondence: L.  S. McDaniel, PhD,  Department of Microbiology and Immunology, University of Mississippi Medical Center,  2500 N State Street, Jackson, MS 39216 (lmcdan- iel@umc.edu). Biofilm Formation Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases A 24-well plate was seeded with 10 colony forming units (CFU) Society of America. pneumococci in biofilm media (Todd-Hewitt broth with 0.5% This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which yeast extract, 8 U/mL catalase, and 10% horse serum) per well and permits non-commercial reproduction and distribution of the work, in any medium, provided the incubated at 37°C with 5% CO for 24 hours. Following incuba- original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com tion, biofilm media was removed from the wells, and biofilms were BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Table 1. Clinical Correlation and Characteristics of NESp Isolated From Patients CA vs Virulence CI Source Age/Sex Presentation/Comorbidity Immunization HA Antibiotic Resistance Gene Encoded C144.66 Middle ear 2/M Chronic adenoiditis ND CA Erythromycin pspK Penicillin Sulfamethoxazole/trimethoprim 361.67 Pulmonary 48/M Intubation PPSV HA Clindamycin pspK ARF 2015 Tetracycline Erythromycin 327.251 Pulmonary 66/M COPD PPSV CA Penicillin aliD Diabetes 2013 and 2016 264.174 Pulmonary 5/F Cystic fibrosis Not immunized CA Erythromycin aliD Abbreviations: ARF, acute respiratory failure; CA, community-acquired; CI, clinical isolate; COPD, chronic obstructive pulmonary disease; HA, hospital-acquired; ND, not determined; NESp, nonencapsulated Streptococcus pneumoniae; PPSV, pneumococcal polysaccharide vaccine (Pneumovax23). stained with 350 µL of 0.1% crystal violet at room temperature for pspK whereas 327.251 and 264.174 encoded aliD (Table 1). All 3 30 minutes. Unbound crystal violet was carefully removed, 1 mL newly identified NESp isolates were associated with pulmonary of 100% ethanol was added to each well to solubilize biofilm-as- disease (Table 1); 361.67 was associated with acute pneumonia sociated crystal violet, and the plate was shaken at room tempera- in an adult male following intubation during acute respiratory ture for 10 minutes. Solubilized crystal violet was transferred to a failure, and 327.251 was isolated during a COPD exacerbation 96-well plate that was measured at OD with an xMark microplate in a 66-year-old male. Both adult male patients were previ- spectrophotometer (BioRad). All samples were assayed in tripli- ously vaccinated against pneumococcus (Table 1); 264.174 was cate, and independent experiments were performed twice. isolated from a cystic fibrosis (CF) pediatric patient who had received no prior vaccination against pneumococcus. The “gold Pneumococcal Adherence and Invasion of Epithelial Cells standard” treatment for S.  pneumoniae infections is penicillin, Human Detroit 562 pharyngeal and A549 pulmonary epithe- and 2 out of the 4 strains were found to be resistant to penicillin lial cells were grown to approximately 90% confluence in 10% (Table  1). NESp strains were also resistant to antibiotics com- fetal calf serum (FCS)–supplemented Eagle’s minimal essential monly used to treat S. pneumoniae infections, such as erythro- medium (EMEM) with 100 µg/mL penicillin, 50 µg/mL strepto- mycin and sulfamethoxazole/trimethoprim. mycin, and 100 µg/mL amphotericin B in 24-well plates at 37°C NESp Phenotypes Permit Persistence in a Host with 5% CO . Pneumococcal adherence and invasion assays PspK is an adhesin that aids in adherence to host cells, and AliC were performed as previously described [11]. Briefly, 10 pneu- and AliD are oligopeptide binding proteins that sense the extra- mococci suspended in antibiotic-free EMEM was added to each cellular environment [5]. We grouped isolates based on expres- well. Adhered pneumococci were enumerated after 30 minutes sion of PspK or AliD to examine virulence mechanisms. Human of incubation with human epithelial cells. To assess invasion, pharyngeal and pulmonary epithelial cells were used to mimic pneumococci were incubated with epithelial cells for 2 hours, the host niche during colonization and pneumonia (Figure 1). All followed by elimination of extracellular bacteria with 200 µg/mL clinical isolates efficiently adhered to pharyngeal cells (Figure 1A). gentamycin before lysing epithelial cells and enumerating intra- PspK-expressing 361.67 adhered to A549 pulmonary cells signif- cellular pneumococcal CFUs. Two independent experiments icantly more (P < .001) than all other isolates (Figure 1B). PspK- were performed, and all samples were assayed in triplicate. expressing cells invaded both pharyngeal and pulmonary cells Statistical Analysis significantly more (P < .001) than their AliD-expressing counter- PRISM 5 software (GraphPad Software, Inc.) was used to analyze parts (Figure 1C and D). When comparing epithelial cell invasion the results. To assess if there were any significant differences in by PspK-expressing strains C144.66 and 361.67, C144.66 demon- the means between all groups compared, we used one-way ana- strated significantly greater (P < .01) invasion of pharyngeal cells lysis of variance (ANOVA). Tukey post-tests allowed us to iden- (Figure 1C), whereas 361.67 demonstrated significantly greater (P tify the specific significant differences in the groups analyzed. < .05) invasion of pulmonary cells (Figure 1D). All NESp strains A P value of less than .05 was considered statistically significant. formed dense biofilms (Figure 1D), but the AliD-expressing strain 327.251 formed significantly more biofilm than the other pulmo- RESULTS nary isolates 361.67 (P < .05) and 264.174 (P < .01). Survey of Pneumococcal Isolates and Clinical Correlation DISCUSSION PCR analysis of the capsule locus demonstrated that 3/35 (~9%) pneumococcal isolates lacked the highly conserved capsule In this report, we determined that nearly 9% of pneumococcal gene cpsA. Further PCR analysis revealed that 361.67 encoded infections at UMMC were linked to NESp, and we described 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 AB Detroit 562 adhesion A549 adhesion 5.5 5.5 *** 5.0 5.0 4.5 4.5 4.0 4.0 3.5 3.5 C144.66 361.67 327.251 264.174 C144.66 361.67 327.251 264.174 pspK aliD pspK aliD C Detroit 562 invasion D A549 invasion *** 3.0 3.0 ** *** *** 2.5 2.5 *** 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 C144.66 361.67 327.251 264.174 C144.66 361.67 327.251 264.174 pspK aliD pspK aliD NESp biofilm formation 0.18 ** 0.16 0.14 0.12 0.10 0.08 0.06 C144.66 361.67 327.251 264.174 pspK aliD Figure 1. Nonencapsulated Streptococcus pneumoniae (NESp) phenotypes influencing pulmonary disease. Human Detroit 562 pharyngeal (A and C) and A549 pulmonary (B and D) epithelial cells were used to assess NESp adherence (A and B) and invasion (C and D). Biofilms formed (E) after 24-hour growth in 24-well plates were assayed using crystal violet staining and measuring the optical density at 630 nm. A, All clinical isolates efficiently adhered to pharyngeal cells. B, Clinical isolate (CI) 361.67 adhered to pulmonary cells significantly more than all other isolates. PspK-expressing cells invaded both pharyngeal (C) and pulmonary (D) cells at low rates. CI C144.66 invaded pharyngeal cells more efficiently than 361.67 (C), whereas 361.67 invaded pulmonary cells more efficiently than C144.66 (D). CI 327.251 formed significantly more biofilm than * ** *** 361.67 or 264.174. Data represent 2 independent studies performed in triplicate. Error bars denote standard error of the mean ( P < .05; P < .01; P < .001). Abbreviations: CFU, colony forming units; OD, optical density. 3 patients who develop NESp-associated pneumonia. We also how NESp are able to establish disease, we examined virulence demonstrated that NESp express antibiotic-resistant phenotypes mechanisms of the clinical isolates. All NESp isolates efficiently that threaten classical treatment measures. As there are no cur- adhered to pharyngeal cells and formed dense biofilms, which rently licensed NESp preventatives, antibiotic-resistant NESp likely aided in nasopharyngeal colonization and development are an emerging threat to public health. To better understand of pneumonia. NESp did not invade epithelial cells efficiently, NESp Associated Bacterial Pneumonia • OFID • 3 OD 630 nm Log CFU/mL Log CFU/mL Log CFU/mL Log CFU/mL Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy135/5034858 by Ed 'DeepDyve' Gillespie user on 16 October 2019 which suggests that NESp are able to withstand the extracellu- risk of diseases associated with these strains. Further surveil- lar environment despite the lack of a protective polysaccharide lance of NESp isolates will need to be conducted to determine capsule. the widespread NESp threat to public health and NESp-specific In the patient infected with 361.67, translocation of 361.67 virulence mechanisms. to the lungs during intubation could have led to the infection, which is a phenomenon that has been previously reported [12]. Acknowledgments As PspK has been shown to increase adherence to pulmonary Financial support. This work was supported by the Medical Scholars Research Program at the University of Mississippi Medical Center and insti- epithelial cells and persistence in a murine lung model, PspK of tutional research funds. 361.67 may have also facilitated persistence during pulmonary Coni fl cts of interest. 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Open Forum Infectious DiseasesOxford University Press

Published: Jul 1, 2018

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