In this assignment, students will review a microbial journal article.
Choose one article from a primary scientific literature source that uses a microbe as a model organism/system and write a comprehensive summary of the study that answers the following questions:
Choose one article from a primary scientific literature source that uses a microbe as a model organism/system.
Write a summary of the study that includes:
- Why did the scientists perform the study (i.e., description of background)?
- What was the hypothesis (or hypotheses) under investigation?
- What were the major results and did they support or negate the hypothesis? Which key techniques were used to achieve these results?
- Why are the results significant and do they point to further/future studies? In other words, why does this article matter and what should or could be done next?
- Why did you choose this particular article to review? Was it interesting, informative, clearly written, or none of the above?
Compose your review in current APA Style and include:
- A title page
- Answers to the questions above in paragraph format (2 or more pages)
- A reference page with the reference for your article and any other sources used in your review.
Article attached.
Molecular characterization of penicillin non-susceptible Streptococcus pneumoniae isolated before and after pneumococcal conjugate vaccine implementation in Casablanca, Morocco Diawara et al. Ann Clin Microbiol Antimicrob (2017) 16:23 DOI 10.1186/s12941-017-0200-6 RESEARCH Molecular characterization of penicillin non-susceptible Streptococcus pneumoniae isolated before and after pneumococcal conjugate vaccine implementation in Casablanca, Morocco Idrissa Diawara1,2*, Abouddihaj Barguigua3, Khalid Katfy1,2, Kaotar Nayme1,4, Houria Belabbes1,2, Mohammed Timinouni4, Khalid Zerouali1,2 and Naima Elmdaghri1,2 Abstract Background: Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, especially among children and the elderly. The ability to effectively treat pneumococcal infection has been compromised due to the acquisition of antibiotic resistance, particularly to β-lactam drugs. This study aimed to describe the prevalence and molecular evolution of penicillin non-susceptible S. pneumoniae (PNSP) isolated from invasive diseases before and after pneumococcal conjugate vaccine implementation in Casablanca, Morocco. Methods: Isolates were obtained from the Microbiology Laboratory of Ibn Rochd University Hospital Centre of Casablanca. Serogrouping was done by Pneumotest Kit and serotyping by the Quellung capsular swelling. Antibiotic susceptibility pattern was determined by disk diffusion and E-test methods. The PNSP were analyzed by pulsed-field gel electrophoresis (PFGE) and by genotyping of pbp1a, pbp2b, and pbp2x genes. Results: A total of 361 S. pneumoniae isolates were collected from 2007 to 2014. Of these isolates, 58.7% were obtained before vaccination (2007–2010) and 41.3% after vaccination (2011–2014). Of the 361 isolates, 80 were PNSP (22.2%). Generally, the proportion of PNSP between pre- and post-vaccination periods were 31 and 13% (p = 0.009), respectively. The proportion of PNSP isolated from pediatric and adult (age > 14 years) patients decreased from 34.5 to 22.9% (p = 0.1) and from 17.7 to 10.2% (p = 0.1) before and after vaccine implementation, respectively. The leading serotypes of PNSP were 14 (33 vs. 57%) and 19A (18 vs. 14%) before and after vaccination among children. For adults, serotypes 19A (53%) and 23F (24%) were the dominant serotypes in the pre-vaccination period, while serotype 14 (22%) was the most prevalent after vaccination. There were 21 pbp genotypes in the pre-vaccination period vs. 12 for post-vaccination period. PFGE clustering showed six clusters of PNSP grouped into three clusters specific to pre- vaccination period (clusters I, II and III), two clusters specific to post-period (clusters V and VI) and a cluster (IV) that contained clones belonging to the two periods of vaccination. Conclusion: Our observations demonstrate a high degree of genetic diversity among PNSP. Genetic clustering among PNSP strains showed that they spread mainly by a restricted number of PNSP clones with vaccine serotypes. PFGE clustering combined with pbp genotyping revealed that vaccination can change the population structure of PNSP. © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Open Access Annals of Clinical Microbiology and Antimicrobials *Correspondence:
[email protected] 1 Laboratoire de Microbiologie, Faculté de Médecine et de Pharmacie, Hassan II University of Casablanca, B.P 5696, Casablanca, Morocco Full list of author information is available at the end of the article http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/ http://creativecommons.org/publicdomain/zero/1.0/ http://crossmark.crossref.org/dialog/?doi=10.1186/s12941-017-0200-6&domain=pdf Page 2 of 9Diawara et al. Ann Clin Microbiol Antimicrob (2017) 16:23 Background Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, especially among children and the elderly. Pneumococcal infections include serious dis- eases such as meningitis, bacteraemia, and pneumonia, as well as milder but more common illnesses, such as sinusitis and otitis media [1]. Disease rates and mortality are higher in developing than in industrialized settings, with the majority of deaths occurring in Africa and Asia. The ability to effectively treat pneumococcal infection has been compromised due to the acquisition of antibi- otic resistance, particularly to β-lactam drugs [2]. Furthermore, antibiotic pressure, in combination with these horizontal recombination events, allows the acqui- sition of antibiotic-resistant genes or resistant strains which increases the resistance to a variety of antibiot- ics [3]. Pneumococcal resistance to β-lactams has been attributed to alterations of the penicillin-binding proteins (PBP) which reduce their affinity [4]. The first pneumo- coccal isolate resistant to penicillin was reported in 1967 from a patient in Australia [5], and resistant pneumococci have subsequently increased in prevalence worldwide. β-Lactam antibiotics exert their biological effects by interacting with the PBPs. PBPs are membrane enzymes that catalyze the polymerization and transpeptidation of glycan strands, during the assembly of the bacterial cell wall. β-Lactam resistance in clinical pneumococci is mediated by altered PBPs, specifically PBP1a, PBP2x and PBP2b [6, 7]. Penicillin resistance in S. pneumoniae is mediated by stepwise alterations of PBPs [8–10]. These three PBPs are considered to be the key targets for these agents and were therefore chosen for examination in this study. In Morocco, the PCV-13 was introduced in the national immunization program in October 2010 in 2 + 1 sched- ule and replaced by the PCV-10 in July 2012. Before pneumococcal vaccine implementation in Morocco, the incidence rate of invasive pneumococcal diseases (IPD) in children aged to 2 years was 34.6/100,000 popula- tions. The incidence rates of PCV-7, PCV-10 non-PCV-7 and PCV-13 non-PCV-10 serotypes were 18.0, 5.7 and 5.7/100,000 population in the same age, respectively [11]. In 2010, the use of the pneumococcal conjugate vac- cine (PCV-13 and then PCV-10) dramatically reduced the prevalence of vaccine serotypes through active vac- cination particularly among children less than 5 years of ages in Casablanca, Morocco. However, the introduction of vaccination was associated with a subsequent rela- tive increase in non-vaccine serotypes [11]. This can be attributed to the phenomena of “serotype replacement”, the expansion of preexisting NVT pneumococci, and/ or serotype switching [12]. Vaccination has also reduced the incidence rate of antibiotic resistant serotypes, but we previously reported, a rebound due to the persistence of some vaccine serotypes like 6B and 14 [11]. Although several studies have described the genetic profile of the pbp1a, pbp2b and pbp2x genes in pneumococci from dif- ferent countries [4, 13], actually, there are no studies on the characteristics of penicillin non- susceptible S. pneu- moniae (PNSP) in Morocco. This study aimed to describe the molecular evolution of penicillin non-susceptible S. pneumoniae isolated from invasive diseases before and after pneumococcal conju- gate vaccine implementation in Morocco in 2010. Methods Bacterial strains, growth conditions and DNA extraction Isolates, collected from 2007 to 2014, were obtained from the Microbiology Laboratory of Ibn Rochd University Hospital Centre of Casablanca (IR-UHC). All the non- duplicate invasive S. pneumoniae isolates recovered from patients, all ages included, during the study periods were included. Isolates obtained from normally sterile sites [cerebrospinal fluid (CSF), blood, pleural fluids, articular fluids or any other sterile site] were considered invasive. The isolates were identified based on the typical colony morphology, Gram staining, optochin sensitivity test (Oxoid Company, Britain) on Mueller–Hinton agar plates supplemented with 5% sheep blood (BioMèrieux, Lyon, France) and bile solubility. The procedures employed for capsular typing and DNA extraction were previously described [14]. Antimicrobial susceptibility Antibiotic susceptibility testing was done following Clinical Laboratory Standard Institute guidelines (CLSI, 2014). Erythromycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole (cotrimoxazole), were tested by disk diffusion with antibiotic disks from Oxoid (Basingstoke, United Kingdom) on Mueller-Hinton Agar supplemented with 5% sheep blood (BioMèrieux, Lyon, France). A minimal inhibitory concentration (MIC) for penicillin G and ceftriaxone was determined on 5% sheep blood Mueller-Hinton agar with E-tests from Oxoid Keywords: Streptococcus pneumoniae, Invasive pneumococcal disease, Penicillin-binding proteins, β-lactams, Serotypes, Antibiotic resistance, PFGE Page 3 of 9Diawara et al. Ann Clin Microbiol Antimicrob (2017) 16:23 (Oxoid, Basingstoke, UK). The breakpoints used for interpretation were those recommended by the CLSI in 2014. Quality control was conducted using S. pneumo- niae ATCC 49619. PCR‑ RFLP of pbp genes Genetic polymorphism of the penicillin resistance genes pbp1a, pbp2b, and pbp2x of the penicillin-nonsusceptible isolates was investigated by restriction fragment length polymorphism (RFLP) analysis as described previously [15]. Briefly, we amplified a segment of 2.4, 1.5, and 2 kb of pbp1a, pbp2b, and pbp2x genes respectively by PCR. PCR amplifications were performed in simplex in a 25 μL reaction mixture containing 0.5 mM of dNTPs, 0.3 μM of each primer, 1× of PCR buffer, 2.5 mM of MgCl2, 1U of Platinum Taq DNA polymerase (Invitrogen). The PCR cycle was 95 °C for 4 min followed by 30 amplification cycles of 94 °C for 1 min, 58 °C (pbp1a), 55 °C (pbp2b) and 60 °C (pbp2x) for 2 min, and 72 °C for 3 min; the final extension was 72 °C for 10 min. The amplification prod- ucts were digested by restriction endonuclease HaeIII and RsaI and separated by agarose gel electrophoresis. Gels were scanned and analyzed by the Geldoc system (Bio-Rad). The different pbp genotypes received a three numbers code (e.g., x/y/z) referring to the RFLP patterns of the genes pbp1a (x), pbp2b (y), and pbp2x (z), respec- tively. As positive control for the three genes, we used 15 penicillin-susceptible S. pneumoniae (PSSP). Pfge PFGE was performed to determine the genetic related- ness among the same pbp genotypes of pneumococcus strains isolated before and after vaccination, following a standardized protocol developed by Bean et al. [16], Elliot et al. [17] and according to the recommendations of the pneumococcal molecular epidemiology network (PMEN). The gel images were processed and analyzed by BioNumerics Ver. 7.5 software (Applied Maths, Belgium). The images were normalized by use of standard molecu- lar markers, and banding patterns were compared. Simi- larity analysis was performed using Dice coefficients and isolates were separated into similarity clusters by the unweighted-pair group method using average linkages (UPGMA). Statistical analysis Data were analyzed with WHONET5.6, EpiInfo 7 (Cent- ers for Disease Control