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ORIGINAL ARTICLE  
Year : 2015  |  Volume : 58  |  Issue : 1  |  Page : 45-47
Selectivity evaluation of a new chromogenic medium to detect group B Streptococcus


1 Department of Diagnostic Services, O.U. Laboratory Analysis of Clinical Chemistry and Microbiology, S. Maria Della Scaletta Hospital, Imola, Via Montericco, 40026 Bologna, Italy
2 Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, Via Pansini, 80131 Naples, Italy

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Date of Web Publication11-Feb-2015
 

   Abstract 

Background: Group B Streptococcus (GBS) is an important pathogen that causes serious infections in newborns. Pregnant screening and intrapartum antibiotic prophylaxis are actually the strategies to prevent GBS disease in neonates because vaccination is under investigation. Materials and Methods: Simultaneously, 156 isolates of GBS and 156 isolates other than GBS covering 17 different species, were tested to evaluate the selectivity of a new chromogenic medium to screen GBS. Results: The new new chromogenic medium showed an excellent performance, exhibiting a very high level of inclusivity (100%) and exclusivity (96.1%).

Keywords: Chromogenic medium, culture, group B Streptococcus, selectivity

How to cite this article:
Pignanelli S, Pulcrano G, Schiavone P, Di Santo A, Zaccherini P. Selectivity evaluation of a new chromogenic medium to detect group B Streptococcus. Indian J Pathol Microbiol 2015;58:45-7

How to cite this URL:
Pignanelli S, Pulcrano G, Schiavone P, Di Santo A, Zaccherini P. Selectivity evaluation of a new chromogenic medium to detect group B Streptococcus. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Jul 5];58:45-7. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/45/151186



   Introduction Top


Group B Streptococcus (GBS) is a major cause of perinatal bacterial infections in pregnant women and focal and systemic infections in newborns from birth until 3 months of age. [1] GBS colonizes the gastrointestinal or genitourinary tracts in 10-30% of pregnant women. [2],[3],[4],[5] Therefore, the neonatal infection results from vertical transmission of GBS. Although screening and intrapartum antibiotic prophylaxis have substantially decreased the incidence of GBS disease in neonates, GBS remains a frequent cause of neonatal morbidity and mortality. [6] In fact, GBS causes systemic infections in elderly people with chronic medical illness. [1] The highest GBS mortality and morbidity result from invasive infections (sepsis and meningitis) in neonates with very low-birth weight. [7] Actually, screening of pregnant women requires a specific culture procedure with an incubation for up to 48 h, precluding intrapartum screening. [6] Another potential strategy for GBS prevention is the introduction of a GBS vaccine, which is still under investigation. [8] The goal of this study was to evaluate the selectivity of a new chromogenic medium versus a comparator to screen GBS in order to easly detect the target colonies.


   Materials and Methods Top


Simultaneously, 156 isolates of GBS and 156 isolates other than GBS covering 17 different species, were tested to evaluate the selectivity of a new GBS screening Brilliance GBS Agar (BGA) medium (Thermo Fisher Scientific, Basingstoke, United Kingdom), alongside ChromidID StreptoB Agar (CSA) (bioMérieux, Marcy-l'Étoile, France). The mixed culture was prepared. It consisted of a suspension with one GBS isolates and one species other than GBS. The mixed culture was prepared in sodium chloride solution (CareFusion, Yorba Linda, USA), with 10 3 CFU/mL GBS and 10 6 CFU/mL species other than GBS inocula. The chromogenic plates were inoculated and incubated according to manufacturer's instructions. After 20 h of incubation, colony color, size, and amount of growth recorded and interpreted according manufacturer's package insert. The species other than GBS were as follows: Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, Listeria monocytogenes,  Salmonella More Details enterica serotype typhimurium, Stenotrophomonas maltophilia, and Candida albicans; for each one six strains were tested. These bacteria showed wild-type antimicrobial profile. In addition, we had selected following pathogenic agents: Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Enterobacter aerogenes,  Escherichia More Details coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, and Pseudomonas aeruginosa; for each one twelve strains were tested. Unlike S. saprophyticus, S. pneumoniae, S. pyogenes, L. monocytogenes, S. enterica, S. maltophilia, and C. albicans isolates, the others were selected as follows: 50% wild type strains, while the remaining strains showed the following resistance: methicillin-resistant S. aureus and with reduced susceptibility to glicopeptides, E. faecalis with reduced susceptibility to glicopeptides, E. faecium glicopeptides resistant, E. aerogenes producing extended-spectrum beta-lactamase (ESβL) and/or carbapenemase, E. coli producing ESβL and/or carbapenemase, K. pneumoniae producing ESβL and/or carbapenemase and/or with multiple drug resistant (MDR) profile, P. mirabilis producing ESβL, A. baumannii and P. aeruginosa with MDR antimicrobial panel. The identification of microorganism and the antimicrobial susceptibility test were performed by the Vitek-2 system and confirmed with disk diffusion tests, as described previously, [9] according to the 2013 European Committee on Antimicrobial Susceptibility Testing guidelines. Moreover, to evaluate the limit of detection (LOD) of the two chromogenic media, inocula of 10 2 UFC/mL, 10 UFC/mL and 1 UFC/mL of GBS were, respectively, added in each medium in triplicate. In addition, as growth control, all the mixed suspensions were inoculated in Sheep Blood Agar (SBA) plates (Bekton Dickinson, Erembodegem, Belgium) and incubated in ambient air at 35°C ± 1°C for 20 h. Any strain demonstrating typical colonial referable to GBS was subcultured into SBA plates (Bekton Dickinson), incubated in ambient air at 35°C ± 1°C for 20 h and then tested with specific Group B latex reagent, according to manifacturer's package insert of Slidex Strepto Plus kit (bioMérieux, Marcy-l'Étoile, France). Furthermore, any colony was identified using biochemical automated test, using GP cards (bioMérieux, Marcy-l'Étoile, France), according to manufacturer's instructions.

The software and criteria used to perform statistical analysis were described previously. [10] Quality controls (QC) for identifications and antimicrobial assays were used; QC strains employed for the identification were: Enterobacter cloaceae ATCC 700323, S. maltophilia ATCC 17666, Enterococcus casseliflavus ATCC 700327, Streptococcus thermophilus ATCC 19258, and C. albicans ATCC 14053 (bioMérieux, Marcy-l'Étoile, France); whereas for the antimicrobial susceptibility test were used E. coli ATCC 25922, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, Candida kruzei ATCC 6258 (bioMérieux, Marcy-l'Étoile, France).


   Results Top


In all tests performed, GBS was detected in both chromogenic media, exhibiting in all cases typical colonies color, showing a high inclusivity (P < 0.001). The colonies isolated into media were red on CSA (bioMérieux), and pink on BGA medium (Thermo Fisher Scientific). [Table 1] displays that the BGA medium (Thermo Fisher Scientific) tested with mixed cultures showed excellent performance (P < 0.001), inhibiting the growth of isolates other than GBS, except for L. monocytogenes. Consequently, 150/156 strains other than GBS were not revealed. The CSA (bioMérieux) medium was less selective, showing mixed colonial growth in 66/156 tests [Table 1], consequently with a more difficult and slow identification. The CSA (bioMérieux) was highly selective for Staphylococcus spp., Streptococcus spp., S. maltophilia, and C. albicans, while not for Enterococcus spp. and L. monocytogenes. The growth of other bacteria into CSA was strictly linked to the resistance mechanisms (P < 0.001), contrary to the BGA (Thermo Fisher Scientific). In addition, LOD for the BGA (Thermo Fisher Scientific) was 1 UFC/mL while for CSA (bioMérieux) was 10 UFC/mL. The SBA plates (Bekton Dickinson) revealed the growth of SBG and another strain other than SGB in all tests. The chromogenic media showed both hemolytic and not hemolytic colonies of GBS. In fact, The SBA plates (Bekton Dickinson) showed that 5 of 156 isolates of GBS were not hemolytic. These colonies exhibited typical coloration into chromogenic media, specific reaction with Group B latex reagent (bioMérieux) and finally, was excellently identified with automated biochemical system (bioMérieux).
Table 1: Growth of strains other than SGB grouped to family


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   Discussion Top


In summary, GBS is an important pathogen that causes serious infections in newborns, pregnant women, and elderly people with chronic illness. [11] Despite the introduction of rapid molecular methods, such as real-time polymerase chain reaction, the cost and required expertise limit their use. [1] In this study, the chromogenic media represent the best choice to screen antenatal GBS for its good performance. Unfortunately, traditional GBS screening methods require a lengthy and often cumbersome preenrichment step. The data obtained on the selectivity of BGA (Thermo Fisher Scientific) permit skipping this step. Subsequently, the workflow is streamlined, and turnaround times are faster. In addition, this approach is suitable for automatic diagnostic methodologies. BGA (Thermo Fisher Scientific) seemed for excellent selectivity an easy-to-read GBS screening medium to provide rapid and accurate results within 20 h based on its innovative inhibigen technology. This technology works by targeting organism-specific enzymatic reactions through the uptake and cleavage inhibitory agents leading to cell lysis. The result is a significant reduction in non-GBS growth on the plate. Unlike conventional selective agents, such as antibiotics, the inhibitory activity applied to the solid medium has resulted independent of the mechanism of bacterial resistance; in fact, it was equally effective to inhibit the growth of wild-type and not wild-type strains other than GBS. In addition, BGA (Thermo Fisher Scientific) showed a very high level of exclusivity, inhibiting the 96.1% of non-GBS isolates growth. Moreover this chromogenic medium, unlike other culture media described in the literature, [12] is able to detect hemolytic and not hemolytic colonies of GBS. Furthermore, the ability to detect L. monocytogenes represent an advantage, because simultaneously it allows to detect two important pathogens during pregnancy. Finally, the detection of the two bacteria is very simple because the color colonies of L. monocytogenes (blue-gray) are easily distinguishable from those of SGB (pink).

 
   References Top

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Kimura K, Nishiyama Y, Shimizu S, Wachino J, Matsui M, Suzuki S, et al. Screening for group B streptococci with reduced penicillin susceptibility in clinical isolates obtained between 1977 and 2005. Jpn J Infect Dis 2013;66:222-5.  Back to cited text no. 1
    
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Correspondence Address:
Dr. Salvatore Pignanelli
Via Guelfa, No. 30, 40138 Bologna
Italy
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.151186

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