b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 159–166 ht tp://www.bjmicrobiol.com.br/ Medical Microbiology Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase a,b a a Adriano Martison Ferreira , Katheryne Benini Martins , Vanessa Rocha da Silva , c a,b,∗ Alessandro Lia Mondelli , Maria de Lourdes Ribeiro de Souza da Cunha a Universidade Estadual Paulista (UNESP), Botucatu Biosciences Institute, Department of Microbiology and Immunology, Botucatu, SP, Brazil b Universidade Estadual Paulista (UNESP), Botucatu School of Medicine University Hospital, Department of Tropical Diseases, Botucatu, SP, Brazil c Universidade Estadual Paulista (UNESP), Botucatu School of Medicine University Hospital, Department of Internal Medicine, Botucatu, SP, Brazil a r a t i c l e i n f o b s t r a c t Article history: Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most impor- Received 3 July 2015 tant staphylococcal species associated with urinary tract infections. The objective of the Accepted 20 June 2016 present study was to compare and to evaluate the accuracy of four phenotypic methods Available online 11 November 2016 for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains Associate Editor: John Anthony produced a halo with a diameter ≤28 mm (penicillin resistant) and all of them were positive McCulloch for the blaZ gene. Among the 28 susceptible strain (halo ≥29 mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determi- Keywords: nation (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the Urinary tract infection least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method Staphylococcus saprophyticus showed the highest specificity (100%). The present results demonstrated that the zone edge Zone edge test test was the most sensitive phenotypic test for detection of beta-lactamase, although it is Beta-lactamase still not an ideal test to detect this type of resistance since its specificity was low. However, blaZ the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method. © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). ∗ Corresponding author at: Departamento de Microbiologia e Imunologia, Instituto de Biociências, UNESP- Univ Estadual Paulista, Rua Prof. Dr. Plínio Pinto e Silva s/n, Distrito de Rubião Júnior, Botucatu, São Paulo, Brasil. CEP 18618-691. Tel.: +551438800428. E-mail: [email protected] (M.L. Cunha). http://dx.doi.org/10.1016/j.bjm.2016.10.011 1517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 160 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 159–166 (zone edge test). However, little information about the reli- Introduction 21,27 ability of the latter phenomenon is available, especially for coagulase-negative staphylococci. The most common etiological agents involved in urinary 28 In 2012, the CLSI started to recommend the zone edge tract infections (UTIs) include enterobacteria, non-fermenting test. This test was found to be more sensitive than the nitro- Gram-negative bacilli, fungi, enterococci, and staphylococci. cefin method in detecting beta-lactamase production in S. Staphylococcus aureus and Staphylococcus saprophyticus are the aureus when only one test was performed. However, laborato- most common and most important staphylococcal species ries could choose to perform first the nitrocefin test and, if the associated with UTIs; however, other coagulase-negative test were positive, to report the isolate to be beta-lactamase staphylococci have gained importance in recent years. positive or penicillin resistant. If the nitrocefin test were neg- S. saprophyticus is the second most common causative agent ative, the zone edge test should be performed before reporting of acute UTI in the community after Escherichia coli and is iso- an isolate to be penicillin susceptible. 1,2 lated mainly from urine of sexually active young women, In view of the high prevalence of penicillin resistance causing symptoms that are undistinguishable from those among staphylococci, penicillin is rarely used as antistaphy- induced by E. coli. Cases of sepsis and pyelonephritis caused lococcal treatment mainly UTIs, where there are several other 3,4 by this microorganism have also been reported. antimicrobial agents that may be used. However, little infor- The occurrence of penicillin resistance in S. aureus iso- mation about these tests is available for coagulase-negative lates recovered from hospitalized patients has been reported staphylococci and better assessment of the detection of 5,6 shortly after the introduction of penicillin in 1941. At the beta-lactamase production in these species is required. The beginning of the 1970s, resistance to penicillin became com- objective of the present study was to compare and to evalu- mon among nosocomial S. aureus isolates (85–90%) and shortly ate the accuracy of the following four phenotypic methods for thereafter penicillinase-producing strains rapidly spread in the detection of beta-lactamase production in Staphylococcus 7–9 the community. Today, more than 90% of S. aureus strains spp.: appearance of the inhibition zone edge around the peni- 10–12 isolated from humans are resistant to penicillin. cillin G disk, disk diffusion, MIC determination, and nitrocefin Penicillin resistance in staphylococci is mediated by the disk test. Detection of the blaZ gene by the polymerase chain production of a penicillinase encoded by the blaZ gene, reaction (PCR) was used as a reference. which inactivates penicillin by hydrolysis of the beta-lactam 13 ring. Four types of penicillinases (A–D) have been described in S. aureus, all of them belonging to Ambler class A Materials and methods 14,15 beta-lactamases. Penicillinases type A, C and D are usually plasmid encoded, while type B is located on the Strains chromosome.16,17 Penicillin-susceptible staphylococci are susceptible to One hundred one Staphylococcus spp. strains isolated from all beta-lactam antibiotics. Penicillin-resistant, oxacillin- urine samples of patients seen in wards, outpatient clinics, susceptible strains are resistant to penicillinase-labile peni- the emergency department and several health centers of Botu- cillins, but are susceptible to other penicillinase-stable peni- catu and region were used in this study. The isolates were sent cillins, beta-lactam/beta-lactamase inhibitor combinations, to the Laboratory of Microbiology of the University Hospital cephems, and carbapenems. Oxacillin-resistant staphylococci (HC), Botucatu Medical School (FMB), between March 10 and are resistant to all currently available beta-lactam antibiotics, November 14, 2008. The project was approved by the Ethics except for new cephalosporins with anti-MRSA activity (cef- Committee of FMB, UNESP (Of.-416/08-CEP). taroline and ceftobiprole). At present, most staphylococci, either coagulase positive Inclusion and exclusion criteria or coagulase negative, are resistant to penicillin G, as well as 18,19 to penicillin V, ampicillin, amoxicillin, and carbenicillin. Patients of both genders and all ages, whose urine cultures Penicillin susceptibility testing of staphylococcal isolates is were positive for Staphylococcus spp. and considered compati- 5 performed in most laboratories by the agar disk diffusion. ble with UTI, with a colony-forming unit count (CFU) ≥10 per 20 29 The Clinical and Laboratory Standards Institute defines mL urine according to the criteria of Kass, were included in penicillin susceptibility as a minimum inhibitory concen- the study. ≤ ␮ tration (MIC) of penicillin 0.12 g/mL or as the formation Strains isolated from urine catheters, suprapubic punc- 5 ≥ of an inhibition halo 29 mm by the disk diffusion method tures and positive urine cultures containing <10 CFU/mL were (Kirby-Bauer), in combination with a negative result in the excluded. chromogenic cephalosporin assay (nitrocefin disk) for detec- tion of beta-lactamase production. Although the disk diffusion Sample size calculation and nitrocefin tests are accepted as methods for determining penicillin susceptibility, studies suggest that these methods The sample size was calculated using the formula of Fisher 21,22 do not reliably detect beta-lactamase production. 30 and Belle, adopting a 95% confidence interval and precision Other methods that have been proposed to improve of 5% for the expected prevalence of patients with UTI. The the sensitivity of beta-lactamase detection in staphylococci proportion of patients with UTI caused by Staphylococcus spp., 23–26 include the clover-leaf test and observation of the appear- which was 5% in a study conducted at the Laboratory of Micro- ance of the inhibition zone edge around penicillin G disks biology of HC-FMB, was used as a basis. b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 159–166 161 20 Although the sample size calculation indicated 73 isolates resistant, respectively.