2012
Ana Catarina Mesquita
Testes fenotípicos para detetar
Montes Rodrigues
genes de resistência a antibióticos
Phenotypical tests to detect antibiotic resistance
genes
2012
Ana Catarina Mesquita
Testes fenotípicos para detetar
Montes Rodrigues
genes de resistência a antibióticos
Phenotypical tests to detect antibiotic resistance
genes
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biotecnologia Molecular, realizada sob a orientação científica da Doutora Sónia Cristina das Neves Ferreira, Diretora do Departamento de Saúde, Ciência e Educação do Instituto da Educação e Cidadania, Mamarrosa e da Doutora Isabel da Silva Henriques, Professora Auxiliar Convidada do Departamento de Biologia da Universidade de Aveiro.
o júri
presidente Prof. Doutor João Manuel da Costa e Araújo Pereira Coutinho
professor associado com agregação do Departamento de Química da Universidade de Aveiro
Prof. Doutora Isabel da Silva Henriques
Professora auxiliar convidada do Departamento de Biologia da Universidade de Aveiro
Doutora Sónia Cristina das Neves Ferreira
Diretora do Departamento de Saúde, Ciência e Educação do Instituto da Educação e Cidadania, Mamarrosa
Doutora Alexandra Sofia Trindade Rodrigues da Silva Moura
Investigadora em pós-doutoramento do Centro de Estudos do Ambiente e do Mar e do Instituto de Investigação Biomédica de Lérida
agradecimentos Dedico este trabalho aos meus pais e à minha irmã que acreditaram em mim e, em tempo algum, me deixaram esmorecer.
À minha orientadora Doutora Sónia Ferreira pelo apoio e incentivo dado desde o primeiro dia deste trabalho; à Doutora Isabel Henriques pela disponibilidade e pelos conhecimentos partilhados; à Maria João Carvalho e ao Pedro Alves pelo apoio no laboratório; à Ana Bartolomeu e à Susana Mendes pelo incansável trabalho desenvolvido no laboratório; ao Paulo Gonçalves por acreditar em mim e pelo apoio psicológico e profissional; e à família de amigos (Tiago, Sabina, Teresa, Ana e Lurdes) que souberam compreender os momentos de silêncio e ausência e que sempre estiveram disponíveis nos momentos de desalento.
Agradeço à BioMérieux pela oferta das placas de Mueller-Hinton e à Iberlab & Imunoreage, na pessoa do Luís Simões, pelos testes fenotípicos da MAST.
palavras-chave Resistência a antibióticos, testes fenotípicos, testes genéticos,
Enterobacteriaceae, saúde pública
resumo A disseminação da resistência a antibióticos é um grave problema de saúde pública que está a tomar proporções com efeitos difíceis de controlar. Nos últimos anos tem-se verificado que esta disseminação está associada com o fluxo de elementos genéticos móveis, como os integrões, os plasmídeos ou os
transposões. Associados a estas estruturas, surgem frequentemente genes que codificam para beta-lactamases, cuja expressão confere resistência a antibióticos frequentemente usados na prática clínica. A causa desta disseminação ainda não é totalmente conhecida, contudo especula-se que possa estar associada ao uso de antibióticos, muitas vezes excessivo ou indevido.
É um fenómeno atual, o facto de grande parte da população mundial estar
envelhecida. Uma consequência óbvia deste facto, é o aumento de população envelhecida infetada com estirpes resistentes, o que dificulta a caracterização dos
backgrounds genéticos existentes no hospital, visto este tipo de pacientes ter uma afluência ao hospital muito elevada. O recurso a testes fenotípicos, de resposta rápida e barata, em laboratórios hospitalares, torna-se cada vez mais necessário. Tendo por base a crescente necessidade de obtenção de resultados que levem a um tratamento mais direcionado, esta tese tem como objetivo principal a aplicação de testes fenotípicos para a deteção de beta-lactamases. Adicionalmente, foram comparadas metodologias existentes no mercado para a deteção de beta-lactamases de espectro alargado (ESBL) e beta-beta-lactamases do tipo AmpC. Para este efeito, foram recolhidas estirpes de bactérias de Gram-negativo produtoras de beta-lactamases, de diferentes produtos biológicos, e compararam-se diferentes testes fenotípicos. Os resultados obtidos foram posteriormente confirmados por metodologias moleculares, pesquisando genes que codificam para beta-lactamases.
A prevalência de Enterobacteriaceae produtoras de ESBL encontrada neste estudo é de 10% (n=498), que é preocupantemente elevada. Estas bactérias predominam nas mulheres (54%) e em crianças e idosos (num total de 78%). Os resultados obtidos mostraram que os testes fenotípicos que fazem a deteção de beta-lactamases, como o teste CicaBeta (MAST, UK), têm elevada sensibilidade (90%). Com testes fenotípicos que permitem uma identificação do tipo de beta-lactamase, como o MASTDISC Test (MAST, UK) e Etest (AB BioMérieux, Sweden), verificou-se uma sensibilidade de 94% e 96%, respetivamente. Os métodos moleculares confirmaram a presença de uma ESBL em 98% dos isolados, que na maior parte dos casos se verificou, usando metodologias moleculares, ser uma CTX-M-15. Existe uma elevada diversidade genética, tendo sido identificados 16 perfis genéticos diferentes e em 82% das estirpes foi identificado um integrão. A presença de isolados produtores de ESBL tem implicações devastadoras, pondo em causa o tratamento dos pacientes. Assim, a rápida deteção deste tipo de isolados torna-se imperativa, não só para a
implementação de uma terapia adequada, mas também para monitorizar a disseminação de resistência.
keywords antibiotic resistance, phenotypical tests, genetic tests, Enterobacteriaceae, public health
abstract The dissemination of antibiotic resistance is a serious problem, difficult to control and poses at risk public health. Several studies report that this dissemination is associated to mobile genetic elements flux, such as integrons, plasmids or transposons. Associated to these structures, are frequently genes coding for beta-lactamases, which expression confers resistance to antibiotics usually used in the clinic. The cause of this dissemination still is not fully known, however it is
speculated that might be associated to the use and misuse of antibiotics. World population is aging. An obvious consequence of this fact is the increasing number of elderly infected with antibiotic resistant strains that attend to the emergency room (ER). Therefore the use of phenotypical tests, which give a fast and economic answer, in hospital laboratories, becomes increasingly necessary to achieve an effective treatment.
The main goal of this thesis was the evaluation of phenotypical tests to detect extended-spectrum beta-lactamases (ESBL) and AmpC-like beta-lactamases. For this purpose, strains with an ESBL-producer phenotype were collected from different biological products, and the performance of different phenotypical tests was compared. The results obtained were subsequently confirmed by molecular methods.
The prevalence of ESBL-producers Enterobacteriaceae found in this study of 10% (n=498) is concerningly high. These bacteria are prevalent in women (54%) and in the early and in elderly ages (in a total of 78%). The results obtained revealed that phenotypical tests that screen for beta-lactamases, as CicaBeta Test (MAST, UK), have a high sensitivity of 90%. Phenotypical tests that allow the identification of the type of beta-lactamase produced, such as MASTDISC Test (MAST, UK) and Etest (AB BioMérieux, Sweden) exhibited higher sensitivity, 94% and 96%, respectively. The molecular methods confirmed the presence of an ESBL in 98% of the isolates, which in most cases was found to be a CTX-M-15, using molecular methodologies. There is a high genetic diversity, being identified 16 different genetic profiles and in 82% of the strains was identified an integron.
The clinical implications of strains carrying beta-lactamases, especially ESBLs and AmpC, are of enormous relevance and can compromise patient's treatment. Thus, the rapid detection of this type of isolates will allow the implementation of
i
Index
1. Introduction ... 1
1.1 Historical overview of antimicrobials ... 3
1.2 Antibiotics ... 6 1.3 Beta-lactams ... 9 1.3.1 Penicillins ... 10 1.3.2 Cephalosporins ... 11 1.3.3 Carbapenems ... 12 1.3.4 Monobactams ... 13 1.3.5 Beta-lactamase inhibitors ... 13 1.4 Antibiotic resistance ... 14 1.4.1 Beta-lactamases ... 15 1.5 Methodologies ... 20 1.6 Enterobacteriaceae ... 21 2. Objectives ... 23
3. Material and methods ... 27
3.1 Central Hospital characterization ... 29
3.2 Bacterial Strains ... 29
3.3 Phenotypical tests to detect the production of beta-lactamases ... 30
3.3.1 Disc-based tests ... 30
3.3.2 Strip-based tests... 31
3.3.3 E-test (AB BioMérieux, Sweden) ... 32
3.4 Semi-automatic tests ... 33
3.5 Genotypic tests ... 35
iii
3.5.2 Molecular typing of bacterial isolates ... 35
3.5.3 Detection of beta-lactamases-encoding genes... 35
3.5.4 Detection of integrons ... 36
4. Results and Discussion ... 39
4.1 Isolates background ... 41 4.2 Phenotypical results ... 46 4.2.1 WalkAway System ... 46 4.2.2 CicaBeta Test ... 49 4.2.3 MASTDISCS Test ... 50 4.2.4 Etest ... 52
4.2.5 Comparison between results obtained using the Etest and MASTDISCS .... 54
4.3 Molecular detection of beta-lactamases genes ... 56
4.4 Molecular typing results ... 62
5. Conclusions ... 65
5.1 General conclusions ... 67
6. References ... 69
v
List of original publications
This thesis includes results from the following publications:
Rodrigues A., Oliveira H., Pereira H., Henriques I., Ferreira S. (2012). Evaluation of
phenotypic methods to detect ESBL-producing Enterobacteriaceae, in Coimbra, Portugal.
vii
List of Tables
Table 1 – Brief overview of the early years of antibiotics’ history: from the sulfa
antimicrobials to therapy with cephalosporins [from (Zaffiri L et al., 2012)].…….…..…..4
Table 2 – Sub-classes of penicillins: Penicillin G and its derivatives (Courvalin P et al., 2010)……….10
Table 3- Sub-classes of cephalosporins: examples of antibiotics and type of activity (Courvalin P et al., 2010).………....11
Table 4 – Gene, primers sequence, annealing temperature and size of the expected fragments...36
Table 5 – Resistance rates (%) to antimicrobials tested in Walkaway System...47
Table 6 – Results obtained for all the isolates with CicaBeta Test...49
Table 7 – Results obtained with MASTDISCS and Etest for E. coli...55
Table 8 – Results obtained with MASTDISCS and Etest for Klebsiella spp...56
Table 9 – Gene profiles obtained for E. coli isolates; + stands for positive; - stands for negative...57
Table 10 – Gene profiles obtained for Klebsiella spp. isolates; + stands for positive; - stands for negative...58
Table 11 – Prevalence of ESBL genes and integrase genes among ESBL-producing isolates………..58
ix Table 12 – ESBL and integrase genes amplified from isolates that gave negative results with the CicaBeta Test……….59
Table 13 – ESBL and integrase genes amplified from isolates that were negative for the screening with the MASTDISCS Test……….60
Table 14 – ESBL-producers determined in WalkAway System that were inconclusive in Etest to detect AmpC beta-lactamases……….61
Table 15 – Groups of E. coli determined by typing techniques: BOX and ERIC...62
Table 16 – Groups of Klebsiella spp. determined by typing techniques: BOX and ERIC...63
xi
List of Figures
Figure 1 - Timeline of antibiotic deployment and the evolution of antibiotic resistance [from (Clatworthy AE et al., 2007)]...5
Figure 2 - Antimicrobial spectrum of activity [from (Madigan MT et al., 2009)]………....7
Figure 3 – Sites of action and potential mechanisms of bacterial resistance to antimicrobial agents. [from (Mulvey MR and Simor AE, 2009)]….……….….……....8
Figure 4 – Chemical structure of beta-lactams antibiotics [from (Sousa JC, 2001)]………...….9
Figure 5 - Chemical structures of various beta-lactamase inhibitors [from (Wright AJ, 1999)]………...13
Figure 6 – WalkAway System equipment (Siemens, USA)………34
Figure 7 – Example of a MicroScan panel NBC 43 for identification and antibiotic profile for Gram-negative bacteria isolated from other samples than urine (Siemens, USA)…….34
Figure 8 – Distribution of ESBL-producing isolates according to the nature of the product.……….42
Figure 9 – Prevalence of ESBL-producing isolates from urine according to the patient’s gender.………..43
Figure 10 – Distribution of ESBL-producing isolates according to each cohort…….………..….44
Figure 11 – Prevalence of ESBL-producing isolates according to patients age...45
xiii Figure 12 – Example of a negative result obtained in MASTDISC Test (MAST, UK)...50
Figure 13 – Example of a positive result to ESBL-producing organism and negative to AmpC beta-lactamases obtained in MASTDISC Test (B-A = 15 and D-C = 14, D-B = -1 and C-A = 0). When no zone of inhibition is observed, it might be considered the diameter of the disc; for example, in disc A, the zone of inhibition is considered to be of 6mm (MAST, UK)...51
Figure 14 - An example of an inconclusive result for the strip of CT/CTL (cefotaxime/ cefotaxime + clavulanic acid) and for the strip of TZ/TZL (ceftazidime/ ceftazidime + clavulanic acid) (BioMérieux, France)...52
Figure 15 – An example of a negative result for Etest CN/CNI (cefotetan/ cefotetan + cloxacilin) and a positive result for Etest PM/PML (cefepime/ cefepime + clavulanic acid) (BioMérieux, France)...53
Figure 16 – An example of results obtained for Etest: A) a deformation ellipse observed in the Etest CT/CTL (cefotetan/ cefotetan + cloxacillin); B) a “phantom” zone observed in the Etest PM/PML (cefepime/ cefepime + clavulanic acid) (BioMérieux, France)...53
xv List of Abbreviations AMK: Amikacin AMP: Ampicillin CAZ: Ceftazidime CFZ: Cefazoline CIP: Ciprofloxacin
CN/CNI: Cefotetan/ cefotetan + clavulanic acid CT/CTL: Cefotaxime/ cefotaxime + clavulanic acid CTX-M-type: Beta-lactamase cefotaxime-type DNA: Deoxyribonucleic acid
dNTPs: Deoxyribonucleotide triphosphates DOX: Doxycyline
ERIC: Enterobacterial repetitive intergenic consensus sequence ESBL: Extended-spectrum beta-lactamase
GEN: Gentamicin
intI1: Gene encoding class 1 integrases MDR: Multidrug resistance
MIC: Minimal inhibitory concentration
MRSA: Methicillin-resistant Staphylococcus aureus OXA-type: Beta-lactamase oxacillin-type
PBPs: Penicillin- binding- proteins PCR: Polymerase chain reaction
PM/PML: Cefepime/ cefepime + clavulanic acid SHV-type: Beta-lactamase sulhydryl variable-type TEM-type: Beta-lactamase Temoneira-type
TZ/TZL: Ceftazidime/ ceftazidime + clavulanic acid VR: Variable region
1
3 1.1 Historical overview of antimicrobials
For many centuries, infections represented the leading cause to the human death, and were always subject of concern among researchers and clinicians (Zaffiri L et al.). The use of homemade potions to treat small infections and wounds dates back to the era before Christ.
The era of antimicrobials started in 1619, with the successful use of cinchone to treat malaria and of ipecacuanha’s root to treat amoebic dysentery (Camargo EP, 1995; Lee MR, 2008). These extracts and its derivatives (alkaloids, quinine and emetine) were, for several years, the only antimicrobial compounds known.
In 1909, after several years of research work with the goal of achieving the key to reduce the mortality and morbidity rates associated to surgeries and infections, Paul Ehrlich discovered the “magic bullet” for the treatment of syphilis and tripanosomiasis. This arsenical compound, the arsphenamine, later called Salvarsan, was the most used antimicrobial drug until the 1940s (table 1) (Thorburn AL, 1983; Zaffiri L et al., 2012). Although Salvarsan was widely used in treatment of syphilis, it had a considerable risk of side effects. Thus, at the beginning of the 40´s, was substituted by penicillin, the most important drug discovered in the last century (Ward PS, 1981; Warren HS, 1918).
In spite of being used only in the 1940s, the penicillin was discovered by Alexander Fleming in 1928, when he noted that Penicillium notatum was able to produce a substance that inhibited the growth of Staphylococcus aureus (Fleming A, 1929). Fleming had some problems in obtaining considerable amounts of penicillin with a high level of purification. It was with Howard Florey that the clinical use of penicillin became widespread, in the early’s 1940s, when he was able to produce sufficient quantities of penicillin (Florey ME et al., 1943; Hare R, 1982). Despite the major breakthrough in the antibiotic field, it did not take much time until the first penicillin-resistant Staphylococcus aureus was reported (Abraham EP, Chain E, 1940). The enzyme responsible for the resistance to penicillin was studied, years later, and described as TEM-type beta-lactamase (Datta N, Kontomichalou P, 1965; Matthew M, 1979). This group of beta-lactamases, particularly TEM-1, later considered as an AmpC ß-lactamase, is nowadays the most frequent and widespread mechanism of resistance to beta-lactams (Jacoby GA, 2009).
In 1932, Klarer and Mietzsch synthesized Prontosil, a sulfonamidochrysoidine that was metabolized to sulfanilamide (Lesch JE, 2007). But only with the efforts of Gerhard Domagk, that proved the effect of this sulfa antibiotic in a murine model with a systemic infection caused by Streptococcus pyogenes, was this prodrug introduced in the clinic as a sulfonamide with a broad-spectrum activity against Gram-negative and Gram-positive bacteria (Bankston J, 2003).
Table 1 – Brief overview of the early years of antibiotics’ history: from the sulfa antimicrobials to therapy with cephalosporins [from (ZaffiriL et al., 2012)].
In an attempt to discover antibiotics with less toxic effects, Selman Waksman and its collaborator Albert Schatz isolated, in 1943, streptomycin from Streptomyces (Schatz A et
5 al., 1944). This was the first effective drug in the treatment of tuberculosis, which secured the Nobel Prize for Medicine to Waksman in 1952 (Zaffiri L et al., 2012).
In 1945, Giuseppe Brotzu discovered that the fungus Cephalosporium acremonium could inhibit the growth of several Gram-negative bacteria (Salmonella typhi, S. paratyphi,
Brucella melitensis, among others) and also Staphylococcus aureus (Muniz CC et al.,
2007). Edward Abraham and some colleagues made some investigations related to the products found by Brotzu. Among these products they isolated one of the most important antibiotics, the Cephalosporin C, that is stable in the presence of the penicillinase produced by Staphylococcus (Sousa JC, 2001; Zaffiri L et al., 2012). Its antibacterial activity and its structure were published by Abraham and Newton in 1961 (Abraham EP, Newton GGF, 1961).
In the 1960s and later years, several compounds with antibiotic activity were discovered, exponentially increasing the options for therapeutic use. In 1960, was discovered the nalidixic acid, the first quinolone (Lesher GY et al., 1962). In the following years were discovered several antibiotics, as shown in the figure 1, belonging to the beta-lactams, such as oxacillin and cephalothin (in 1962); aminoglycosides, such as gentamicin (in 1963) and tobramycin (in 1968). In the 1970s another breakthrough was achieved with
Figure 1 - Timeline of antibiotic deployment and the evolution of antibiotic resistance [from (Clatworthy AE et al., 2007)].
the discovery of beta-lactamases inhibitor, clavulanic acid (in 1976). In the 1980s, there was an improvement in antimicrobial therapy with the discovery of ceftazidime (in 1980) and fluoroquinolones (in the 1980s) (Sousa JC, 2001).
1.2 Antibiotics
Antibiotics can be defined as substances, natural or synthetic, that in small amounts have activity against bacteria. These substances can be used to treat or prevent infectious diseases in humans or other animals. Antibiotics may either have bactericidal or bacteriostatic effect on the bacteria responsible for the infection, with no toxic effects to the humans or animals. The antibacterial activity of an antibiotic can be measured in vitro by determining the minimal inhibitory concentration (MIC). The main feature of antibiotics is the selective toxicity, which is the ability of selectively act against the microorganism without causing damage or secondary effects on the host. This feature is expressed in terms of therapeutic index, obtained by the relationship between the therapeutic dose (optimal concentration for treatment) and the toxic dose (minimal toxic concentration) (Carneiro M et al., 2011; Madigan MT et al., 2009). Antibiotics of the same class have similar modes of action and can have similar spectra of activity; they generally share resistance and are similar in their toxicity.
There are several classifications of the antibiotics based on their chemical structure, biological origin and their therapeutic use. Therefore, and according to their biological origin antibiotics may be divided, according to their source (Courvalin P et al., 2010), in:
- natural, when they are a secondary metabolic product of bacteria (e.g., Actinomycetes) or filamentous fungi (e.g., Penicillium), such as Penicillin G or when they are extracted from plants;
- synthetic, when they are completely produced in the laboratory, such as quinolones, sulfonamides or chloramphenicol;
- semisynthetic, when natural antibiotics are modified adding chemical groups, which make them less susceptible to inactivation by microorganisms, such as aminopenicillins.
Antibiotic drugs can be classified, based on activity, as “bactericidal”, when they can destroy bacteria, or “bacteriostatic”, when they only suppress bacterial growth, allowing
7 the immune system to resolve the infection (Madigan MT et al, 2009; Scheld WM, Sande MA, 1983; Sousa JC, 2001).
Antibiotics can also be classified according to their spectrum of action. A few antibiotics, which act selectively, having activity against only one group of bacteria, are called narrow spectrum antibiotics. On the other hand, antibiotic that have activity against more than one group of bacteria, are called broad spectrum antibiotics, as represented in figure 2 (Madigan MT et al., 2009).
Figure 2 – Antimicrobial spectrum of activity [from (Madigan MT et al., 2009)].
In addition, most antimicrobial agents used for the treatment of bacterial infections may be classified according to their principal mechanism of action. The mechanisms of action of most antibiotics are well known, and are generally similar in drugs of the same group, giving rise to a pattern of complete cross-resistance.
There are five major mechanisms of action of antibiotics on specific structures or metabolic pathways, allowing a better understanding about antibiotics nature and its selective toxicity (Madigan MT et al., 2009; Sousa JC, 2001; van Hoek AHAM et al., 2011) (figure 3).
Antibiotics act by:
- inhibiting of cell wall synthesis, also known as anti-parietal antibiotics, inhibit the synthesis of peptidoglycan, the polysaccharide that gives structure to the cell wall and protects the bacterial cell from cellular lysis. Are the most selective antimicrobials and present the highest therapeutical index. Belong to this group: fosfomycin, D-cycloserine, bacitracin, vancomycin, teicoplanin and beta-lactams;
- active on the membrane, due to its connection to the bacterial cytoplasmic membrane, changing its permeability. Antibiotics using this mechanism of action have a lower selective toxicity. As examples of antibiotics belonging to this group can be referred: polymyxins and ionophores molecules, as gramicidin A and daptomycin;
- inhibiting the protein synthesis, generally quite selective, is a major group of antibiotics. The protein synthesis is a highly complex process that involves several steps, molecules and structures that may be affected by these antibiotics. Belong to this group: aminoglycosides, like gentamicin and streptomycin; tetracyclines, chloramphenicol, macrolides, as erythromycin; lincosamides and streptogramins;
- inhibiting the nucleic acid synthesis, antibiotics using these mechanisms of action have a variable selectivity. Belong to this group: rifampicin, quinolones and novobiocin;
Figure 3 – Sites of action and potential mechanisms of bacterial resistance to antimicrobial agents. [from (Mulvey MR and Simor AE, 2009)].
9 - altering the metabolic pathways, these antibiotics can inhibit, competitively, the metabolic chain, by having more affinity for enzymes of the metabolic chain than to its natural substrates. Belong to this group: sulphonamides, sulfones and PAS, trimethoprim and isoniazid.
1.3 Beta-lactams
Beta-lactam antibiotics are a very broad class that includes penicillins, cephalosporins, monobactams and carbapenems. All the antibiotics belonging to this class are characterized by having a beta-lactam ring which is indispensable to the antibacterial activity (figure 4) (Darville T, Yamauchi T, 1994; Madigan MT et al., 2009; Poole K, 2004; Sousa JC, 2001).
This class also includes the beta-lactamases inhibitors that, although they have no antimicrobial activity, possess a beta-lactam ring in its chemical structure and may be administrated in combination with beta-lactam antibiotics (Sousa JC, 2001; Wright AJ, 1999).
The beta-lactam antibiotics act in peptidoglycan biosynthesis and have a great affinity to transpeptidases (originating PBPs, Penicillin-Binding-Proteins), acylating them, preventing the occurrence of transpeptidation during peptidoglycan biosynthesis and activating autolytic activity of bacteria (Sousa JC, 2001). For this reason, these antibiotics are bactericidal.
1.3.1 Penicillins
This sub-class of beta-lactams was the first one discovered. With Fleming’s find, the penicillin started the era of antibiotics and the clinical use of beta-lactams. In its structure there is a beta-lactam ring but also a side chain that, due to variations, is responsible for the antibacterial spectrum and for the wide range of derivatives (Wright AJ, 1999) (table 2).
Compared to other sub-classes of antibiotics, the penicillins have a bactericidal activity and have one of the widest spectrums of activity.
The penicillins have a high tissue penetration, although have relatively nontoxic effects and are effective to treat infections caused by a wide range of bacteria, including Gram-negative and Gram-positive, cocci or rods, and some anaerobes (Wright AJ, 1999; Zaffiri L et al., 2012).
Table 2 – Subclasses of penicillins: Penicillin G and its derivatives (Courvalin P et al., 2010).
Subclasses Antibiotics
Natural penicillins or narrow-spectrum
Penicillin G (or benzylpenicillin) Penicillin G procaine Penicillin G benzathine Penicillinase-resistant penicillin or very-narrow spectrum Methicillin Oxacillin Nafcillin Cloxacillin Dicloxacillin Aminopenicillins* Ampicillin Amoxicillin Bacampicillin Carboxypenicillins* Carbenicillin Ticarcillin Ureidopenicillins or broad-spectrum† Mezlocillin Azlocillin Piperacillin
Penicillin plus beta-lactamase inhibitors
Amoxicilin-clavulanic acid Ampicillin-sulbactam Ticarcillin-clavulanic acid Piperacillin-tazobactam * Both sub-classes are considered extended-spectrum penicillins. † This sub-classe is also known as antipseudomonal penicillins.
11 1.3.2 Cephalosporins
Cephalosporins started to be clinically used in the 60’s, with the discover of cephalothin (1962) and cefazolin (1970), both parenteral drugs, and cephalexin (1967), an oral agent, which are considered cephalosporins of first generation (Darville T and Yamauchi T, 1994; Zaffiri L et al., 2012). Until today, more than 20 cephalosporin antibiotics were released for clinical use (table 3).
Table 3- Subclasses of cephalosporins: examples of antibiotics and type of activity (Courvalin P et al., 2010).
Generation Antibiotics Type of activity
Cephalosporins 1st generation Cefadroxil Cefazolin Cephalothin Cephalexin Gram-positive cocci, E. coli and Klebsiella spp.
Cephalosporins 2nd generation Cefaclor Cefamandole Cefoxitin Cefuroxime Cefotetan
Usually more effective than penicillin against Gram-negative bacilli. Haemophilus influenzae, Enterobacter, Neisseria, Proteus, E. coli e Klebsiella spp. Cephalosporins 3rd generation Cefixime Cefoperazone Cefotaxime Cefpodoxime Ceftazidime Ceftriaxone
Generally used to treat infections caused by bacteria resistant to other beta-lactams and also in prophylaxis prior to orthopedic and abdominal surgeries
Cephalosporins 4th generation
Cefepime Cefaclidine Cefpirome
Better against Pseudomonas and Gram-positive bacteria
Cephalosporins 5th generation
Ceftobiprol Ceftaroline
Additional activity against MRSA
The structure of cephalosporin antibiotics is similar to the one of penicillins, although the side chain of beta-lactam ring is the difference that provides to cephalosporins the ability to resist to bacterial enzymes. The possibility of modification of the cephalosporin
nucleus seems to be unlimited, which allows a wide range of activity of this sub-class (Darville T and Yamauchi T, 1994).
The cephalosporins have antibacterial activity, which is variable and affected by several factors, as the ability to resist to the degradation by enzymes and the ability to penetrate the bacterial cell wall (Zaffiri L et al., 2012). However, their activity against penicillin-resistant pneumococci, methicillin-resistant staphylococci or enterococci,
Listeria or Chlamydia is not reliable (Darville T and Yamauchi T, 1994).
1.3.3 Carbapenems
Carbapenems are the beta-lactams with the broadest activity spectrum and act by inhibiting bacterial cell wall synthesis by binding to penicillin-binding proteins (PBPs) and inactivating them (Zhanel GG et al., 2007). This sub-class is stable to most beta-lactamases including AmpC beta-lactamases and extended-spectrum beta-lactamases (ESBL) (Bodey GP, 1990; Zhanel GG, 2007).
In this sub-class are included imipenem, the first carbapenem known, meropenem, doripenem and ertapenem. Carbapenems, as imipenem, meropenem and doripenem, are active against many Gram-positive (streptococci, methicillin-sensitive staphylococci), Gram-negative (Neisseria, Haemophilus and the common aerobic Gram-negative nosocomial pathogens including Pseudomonas) and anaerobic bacteria (Bodey GP, 1990; Hellinger WC, Brewer NS, 1999; Zhanel GG, 2007). However, ertapenem has a more limited spectrum of activity, mainly because it lacks activity against Pseudomonas
aeruginosa and Enterococcus spp (Zhanel GG, 2007). Carbapenems, in general, lack
activity against Enterococcus faecium, methicillin-resistant Staphylococcus aureus and
Stenotrophomonas maltophilia (Hellinger WC, Brewer NS, 1999; Zhanel GG, 2007).
The carbapenems have a low toxicity associated and may be considered for treatment of mixed bacterial infections and aerobic Gram-negative bacteria not susceptible to the other beta-lactams (Hellinger WC, Brewer NS, 1999).
13 1.3.4 Monobactams
Monobactams sub-class is the most recent sub-class of beta-lactam antibiotics and is characterized by a “naked” beta-lactam ring, which allows it a weak antibacterial activity. Aztreonam, the only commercialized monobactam, is a completely synthetic product that is resistant to the action of most lactamases (Bodey GP, 1990). However, several beta-lactamases can inactivate this antibiotic, hydrolysing the beta-lactam ring, such as ESBLs and carbapenemases (Bradford PA, 2001; Nordmann et al., 2011).
Aztreonam is active against most aerobic Gram-negative organisms, including P.
aeruginosa, Haemophilus influenzae and Neisseria spp., and completely inactive against
Gram-positive organisms and anaerobic bacilli. It is an alternative for patients with penicillin or cephalosporin allergies, for combined treatments with aminoglycosides and for the treatment of aerobic Gram-negative infections, being often used in combination therapy for mixed infections (aerobic and anaerobic organisms) (Bodey GP, 1990; Hellinger WC, Brewer NS, 1999).
Monobactams are not nephrotoxic drugs and are not associated with coagulation’s disorders. They also are weakly immunogenic (Hellinger WC, Brewer NS, 1999).
1.3.5 Beta-lactamase inhibitors
Beta-lactamase inhibitors are administered to prevent the inactivation of beta-lactams by beta-lactamases, since they bind irreversibly to the latter. Clavulanic acid, sulbactam and tazobactam are beta-lactam inhibitors (figure 5) and are used associated to beta-lactam antibiotics (Sousa JC, 2001; Wright AJ, 1999). A broadly known example is amoxicilin/clavulanic acid.
1.4 Antibiotic resistance
The dissemination of antibiotic resistance is a global problem mainly associated to health care. With the flow of people, particularly of old-aged people from hospital to households and vice versa, the increase of antibiotic resistance is out of control and became a public health problem with proportions without borders (ECDC, 2011).
The constant use of antibiotics creates selective pressure on the bacteria, allowing the survival of the bacterial population resistant to antibiotics. Bacteria can avoid potentially toxic compounds using single or combined mechanisms of resistance, which may involve different elements. The mechanisms of bacterial resistance may be either intrinsic or acquired. The former is so called intrinsic, when it is a typical feature of the species, which means it is a characteristic displayed by all the members of a given species (Courvalin P et al., 2010). For instance, all the strains of that bacterial species are likewise resistant, or can be potentially resistant to an antibiotic. A classical example of intrinsic resistance is that displayed by the Mycoplasma genus to beta-lactam antibiotics, since the Mycoplasma genus lacks the cell wall, which is the beta-lactam target (Razin S et al., 1998).
On the other hand, the acquired resistance differs from the intrinsic in that the former is not present in all species members but only within a certain lineage of bacteria derived from a susceptible parent (Harbottle H et al., 2006; Tenover FC, 2006). The occurrence of acquired resistance may be due to mutations, exogenous acquisition of DNA (transduction, transformation and conjugation) and intramolecular rearrangements in the DNA (transposons and integrons) (Alekshun MN, Levy SB, 2007; Stokes HW, Gillings MR, 2011).
In an attempt to survive, bacteria have developed biochemical mechanisms of resistance to antibiotics, impairing the binding of the antibiotic to its target. It can be done by several mechanisms, as shown in figure 3, alone or cumulatively, including: a) alterations in the permeability of the bacterial cell wall, preventing the access of antibiotics to the target molecules; b) activation of efflux pumps, removing the antibiotic from the bacterial cell; c) modification or substitution of the antibiotic target, such as PBPs; d) enzymatic modification of the antibiotic; e) overproduction of the target enzyme; f) adoption of alternative metabolic pathways to the ones inhibited by the specific antibiotic;
15 g) inactivation and degradation of the antibiotic by specific enzymes, such as beta-lactamases (Sousa JC, 2001; van Hoek AHAM et al., 2011).
1.4.1 Beta-lactamases
Beta-lactamases are hydrolases (EC 3.5.2.6) produced by negative and Gram-positive bacteria that cleave the amide bond of the beta-lactam ring (Kong KF et al., 2010). Beta-lactamases mostly are serine proteases because they have a serine molecule in the active site (Marsik FJ, Nambiar S, 2011). In case of carbapenemases, beta-lactamases directed to carbapenems, they are mostly metallo-enzymes, due to zinc ions in the active site (Nordmann P et al., 2011; Queenan AM and Bush K, 2007).
Over the years, many classification schemes have been presented, due to the increasing number of enzymes that have been discovered, as well to the approach taken to perform the enzymes division.
Two general schemes of classification were proposed, namely the classification of Ambler, based in the amino acids sequence of four studied beta-lactamases (Ambler RP, 1980; Poole K, 2004), and the classification of Bush, based in biochemical features, as the profile of substrates and inhibitors (Bush K, Jacoby GA, 2010).
In Gram-positive bacteria, these penicillinase-type enzymes are excreted to the outside of the cell, therefore they hydrolyze penicillins more efficiently than cephalosporins (Bush K et al., 1995). Usually, genes encoding this type of enzymes are located in plasmids (Wilke MS et al., 2005). In Gram-negative bacteria, beta-lactamases are more frequent and can not cross the outer membrane, being retained and acting in the bacterial periplasm. This kind of beta-lactamases might be plasmid-encoded or chromosomal-encoded (Livermore DM, 1995; Samaha-Kfoury JN and Araj GF, 2003; Shah AA et al., 2004; Sousa JC, 2001).
As stated above, genes responsible for the production of beta-lactamases might have chromosomal location, such as the ampC gene, or can be located in plasmids or transposons, such as the blaTEM or blaCTX-M genes (Hanson ND and Sanders CC, 1999; Medeiros A, 1997). However, cases have been described in which chromosomal-encoded beta-lactamases from a specific species are plasmid-encoded beta-lactamases in another species. That is the case, for example, of SHV-1 beta-lactamase that is encoded in the
chromosome in Klebsiella pneumoniae strains and plasmidic in E. coli strains (Livermore DM, 1995; Sousa JC, 2001).
1.4.1.1 Extended-Spectrum Beta-Lactamases (ESBL)
Extended-spectrum beta-lactamases (ESBLs) were discovered in 1983 and are usually plasmid-mediated beta-lactamases (Bush K and Jacoby GA, 2010; Livermore DM, 1995; Samaha-Kfoury JN and Araj GF, 2003; Thomson KS, 2001). Most descend by genetic mutation from broad-spectrum beta-lactamases (as TEM-1, TEM-2, SHV-1) found in Gram-negative bacteria, especially E. coli and Klebsiella spp.(Cormican MG et al., 1996; Philippon A et al., 1989).
Plasmid-mediated ESBLs confer resistance to a broad range of beta-lactams, allowing the arise of ESBL-producing strains resistant to the widely variety of antimicrobials commonly used (Cormican MG et al., 1996; Pfaller MA, Segreti J, 2006), which is becoming a global health concern.
These beta-lactamases are able to hydrolyze a wide variety of penicillins, cephalosporins (including third-generation cephalosporins) and monobactams (aztreonam). Nevertheless, they keep the susceptibility to cephamycins (second-generation cephalosporins as cefoxitin and cefotetan) and carbapenems, and are inhibited by clavulanate or other beta-lactamases inhibitors (Bradford PA, 2001; Gniadkowski M, 2001; Pfaller MA and Segreti J, 2006).
According to Ambler’s classification, the majority of ESBLs belong to classes A and D, and they have a serine in their active site. They can hydrolyse oxyimino- beta-lactam compounds at a rate equal or higher than 10% of that for benzylpenicillin (Cormican MG et al., 1996; Gniadkowski M, 2001). ESBLs can also be divided by Bush classification into two subgroups of group 2: subgroup 2be, with extended-spectrum beta-lactamases (Ambler’s class A enzymes), and subgroup 2d, with cloxacillin-hydrolyzing beta-lactamases (Ambler’s class D enzymes) (Bush K and Jacoby GA, 2010; Gniadkowski M, 2001; Paterson DL and Bonomo RA, 2005; Samaha-Kfoury JN and Araj GF, 2003).
At present are known more than 1000 different beta-lactamases (Marsik FJ and Nambiar S, 2011), most of them are ESBL variants that are divided into nine distinct
17 families (TEM, SHV, CTX-M, OXA, PER, VEB, GES, TLA and BES), based on deduced amino acid sequence (Gniadkowski M, 2001). The most common families are TEM, SHV, CTX-M and OXA.
TEM
In this family, TEM-1 is the most common beta-lactamase found in Gram-negative bacteria. Approximately 200 genetic variants descend from TEM-1, (www.lahey.org, accessed in November 2012), most of which confer resistance to ampicillin in E. coli and other bacteria (Bradford PA, 2001; Pfaller MA and Segreti J, 2006; Samaha-Kfoury JN and Araj GF, 2003).
The first TEM described (TEM-1) was discovered in 1965 in E. coli, isolated from a biological product from a girl named Temoneira (hence the designation TEM) (Paterson DL and Bonomo RA, 2005) in Greece, and it spread rapidly to other bacteria. Although TEM-type beta-lactamases are most often found in E. coli and K. pneumoniae, they are also found in other Enterobacteriaceae (such as Enterobacter aerogenes, Morganella
morganii, Proteus mirabilis and Salmonella spp.) and in other penicillin or ampicillin
resistant Gram-negative bacteria (such as Haemophilus influenzae and Neisseria
gonorrhoeae) (Bradford PA, 2001; Samaha-Kfoury JN and Araj GF, 2003).
TEM-1 and TEM-2 (differing in the native promoter and isoelectric point) are able to hydrolyze ampicillin at a greater rate than carbenicillin, oxacillin or cephalothin, and have a very low activity against extended-spectrum cephalosporins. They are inhibited by clavulanic acid (Paterson DL, Bonomo RA, 2005).
The first TEM-type ESBL reported was TEM-3, that was discovered in K. pneumoniae and is a plasmid-mediated beta-lactamase that confers a complex antibiotic resistance phenotype (Pfaller MA and Segreti J, 2006). In the past two decades, several studies were developed to characterize the TEM variants in clinical isolates, among them ESBL TEM-type enzymes, such as TEM-14, TEM-19, TEM-25, TEM-47, TEM-48, TEM-68 and some of them with resistance to the inhibitors (Baraniak A et al., 2005; Mabilat C and Courvalin P, 1990).
SHV
The SHV-type ESBLs derived from SHV-1, which is most commonly found in K.
pneumoniae and confers a high level of resistance against ampicillin (Bradford PA, 2001;
Pfaller MA and Segreti J, 2006). There are over 160 genetic variants of SHV (sulhydryl variable) enzymes (www.lahey.org, accessed in November 2012).
The majority of SHV-type ESBLs are found in K. pneumoniae, but they can also be found in a wide range of Enterobacteriaceae (like Citrobacter diversus, E. coli) and in P.
aeruginosa and Acinetobacter spp. (Bradford PA, 2001; Paterson DL and Bonomo RA,
2005).
CTX-M
CTX-M is a recent family of plasmid-mediated ESBLs and its designation refers to its potential hydrolytic activity against cefotaxime (Bonnet R, 2004; Bradford PA, 2001; Paterson DL and Bonomo RA, 2005; Pfaller MA and Segreti J, 2006). Many CTX-M-type enzymes hydrolyze cefepime as well. These enzymes are inhibited by tazobactam, 10-fold more than by clavulanic acid.
The CTX-M enzymes are not closely related to TEM and SHV-type beta-lactamases but they are related to the chromosome-encoded AmpC-type beta-lactamases of Kluyvera spp. (Paterson DL and Bonomo RA, 2005).
The blaCTX-M has been reported in many species of Enterobacteriaceae and their rapid dissemination usually involves plasmid or epidemic strains (Bradford PA, 2001; Paterson DL and Bonomo RA, 2005; Pfaller MA and Segreti J, 2006).
OXA
The OXA-type enzymes, which are the only ESBLs of class D, or oxacillinases, are characterized by higher hydrolysis rates for cloxacillin and oxacillin than for benzylpenicillin or third-generation cephalosporins (Bradford PA, 2001; Paterson DL and Bonomo RA, 2005; Pfaller MA and Segreti J, 2006). They are poorly inhibited by clavulanic acid, tazobactam and sulbactam, whereas can be inhibited by sodium chloride (Bush K and Jacoby GA, 2010; Poirel L et al., 2010), except OXA-18 that is totally inhibited by clavulanic acid (Pfaller MA and Segreti J, 2006).
19 OXA-type ESBLs are most often found in P. aeruginosa, rather than in
Enterobacteriaceae (Paterson DL and Bonomo RA, 2005; Pfaller MA and Segreti J, 2006).
1.4.1.2 AmpC Beta-Lactamases
Another common mechanism of resistance to beta-lactams in Enterobacteriaceae is the one involving the production of AmpC beta-lactamases, which can be chromosomal or plasmid-mediated (Jacoby GA, 2009; Pfaller MA and Segreti J, 2006; Philippon A et al., 2002). This type of enzymes belong to class C of Ambler’s structural classification and to class 1 of Bush’s functional classification (Ambler RP, 1980; Bush K and Jacoby GA, 2010).
AmpC beta-lactamases are active against aminopenicillins, cephalosporins, oxyimino-cephalosporins (such as ceftriaxone, cefotaxime and ceftazidime), cephamycins (such as cefoxitin and cefotetan) and monobactams (aztreonam) (Jacoby GA, 2009; Peter-Getzlaff S et al., 2011). This broad phenotype of resistance can be due to: a) the overexpression of AmpC beta-lactamases caused by mutations on the repressor gene ampD or in the ampC gene promoter; b) plasmid-encoded AmpC beta-lactamases, or c) induction of chromosomal AmpC beta-lactamases, in response to beta-lactam exposure (Briñas L et al., 2005; Jacoby GA, 2009; Nelson EC and Elisha BG, 1999; Peter-Getzlaff S et al., 2011; Pfaller MA and Segreti J, 2006; Philippon A et al., 2002).
The organisms producing AmpC beta-lactamases share a very similar antimicrobial resistance profile with the ESBL producers. However, unlike the majority of ESBLs, the AmpC enzymes are not inhibited by clavulanate, tazobactam and sulbactam (Peter-Getzlaff S et al., 2011; Pfaller MA and Segreti J, 2006; Philippon A et al., 2002). Nevertheless, some AmpC-type beta-lactamases are inhibited by tazobactam, sulbactam, cloxacillin, oxacillin and/or aztreonam. AmpC beta-lactamases are poorly inhibited by p-chloromercuribenzoate and not at all by EDTA (Jacoby GA, 2009).
K. pneumoniae, Klebsiella oxytoca, P. mirabilis and Salmonella spp. are examples of
bacteria that lack a chromosomal blaAmpC gene. On the other hand, Citrobacter spp.,
chromosomal AmpC beta-lactamases (Jacoby GA, 2009; Philippon A et al., 2002; Thomson KS, 2001).
Although chromosome-encoded AmpC are usually inducible enzymes, expressed at low basal levels in the absence of the selective pressure, in E. coli these enzymes are non-inducible. However, some strains may possess a strong promoter preceding the ampC gene, and therefore express the beta-lactamase gene at high levels (Peter-Getzlaff S et al., 2011; Pfaller MA and Segreti J, 2006; Thomson KS, 2001).
Besides the chromosomal ampC encoding gene, E. coli can carry plasmids with ampC (pAmpC), acquired by horizontal gene transfer and derived from the chromosomal ampC genes from other members of the Enterobacteriaceae family (Jacoby GA, 2009; Peter-Getzlaff S et al., 2011). Among the plasmid-mediated AmpC enzymes, probably derived from chromosomal AmpC enzymes, can be found the following clusters: LAT-types and certain CMY-types of C. freundii group, the Enterobacter group with MIR-1 and ACT-1, the M. morganii group with DHA-1 and DHA-2, the H. alvei group represented by ACC-1, and the Aeromonas group with MOX-, FOX- and other CMY-type enzymes (Jacoby GA, 2009; Philippon A et al., 2002). Plasmid-encoded AmpC beta-lactamases confer resistance to a wide range of antibiotics: penicillins, oxyimino-cephalosporins, cephamycins and monobactams (Jacoby GA, 2009; Philippon A et al., 2002).
1.5 Methodologies
The characterization of the antibiotic resistance phenotype of clinical isolates is very important for establishing a correct and appropriate therapy to the patient and to monitorize the response to the therapeutic treatment. Although, the phenotypical characterization is not immediate nor provides a result in the same day, there are some tests that can provide a result in 16-24 hours after the pathogen isolation. The fast determination of the phenotype of the pathogen and, in the case of beta-lactam resistance, the determination of the type of beta-lactamase produced is crucial for the effectiveness of the treatment, which, in some cases, must be instituted in the first 24 hours after diagnosis.
In fact, the detection and determination of the type of beta-lactamases produced may differentiate the clinical treatment used. If an ESBL is detected, the treatment may include
21 beta-lactams inhibitors, whereas if an AmpC beta-lactamase is identified, then the treatment can not include beta-lactams inhibitors.
The phenotypical characterization can be executed according to several methodologies, including manual or semi-automatic tests. The manual tests, the most traditional ones, still are the reference tests to determine susceptibility/resistance of organisms. Among this type of tests, are the disc tests, the strip tests (screening tests) and E-test (AB BioMérieux, Sweden) that give a resistance pattern approximately 48 hours after the isolation and identification of the organisms. The Walkaway System (Siemens, USA) and Vitek2 (BioMerieux, France) are examples of semi-automatic tests that can give the identification and the phenotypical profile to antibiotics at the same time, 16-24 hours after the isolation of the organisms. Despite being of very rapid execution, they lack some confirmatory tests in case of doubtful results.
1.6 Enterobacteriaceae
Enterobacteriaceae, also known as fermentative bacteria or coliforms, is a very large
family of bacteria, which includes enteric Gram-negative rods. These microorganisms are part of the normal intestinal flora of most animals, including humans. Many of them are pathogens to human health, causing most of intestinal infections as well as urinary and respiratory tracts infections and sepsis (Jarzab A et al., 2011).
To this family belong species, such as E. coli, K. pneumoniae, P. mirabilis,
Enterobacter spp, which are members of the normal flora that may cause opportunistic
infections. Other species, such as Salmonella spp., Serratia spp., Shigella spp., Yersinia spp. are frequently associated to human diseases (Sousa JC and Ferreira WC, 2000).
Enterobacteriaceae are aerobes or facultative anaerobes, all fermenting a wide range of
carbohydrates, such as glucose (used anaerobically). Members of this family are oxidase negative, catalase positive and reduce nitrate to nitrite. Usually are motile, except
Klebsiella and Shigella that are nonmotile. They have antigenic structures that are
important to determine the pathogenicity and virulence of the bacteria (Madigan MT, 2009).
Enterobacteriaceae can survive in many environments, like in the water (Machado E et
al., 2009), animals, plants and are associated to the meat handling (Stiles ME and Lai-King NG, 1981). They are considered a serious problem due to the increasing rates of antibiotic resistance registered among them, because of the transfer of mobile genetic elements that occur between the members of this family (Bush K, 2010; Susic E, 2004).
Among Enterobacteriaceae, E. coli is the most common pathogen identified in urinary tract infections. Although, K. pneumoniae and K. oxytoca are associated to urinary tract infections, they can also cause respiratory tract infections (Sousa JC and Ferreira WC, 2000).
23
25 As stated before, antibiotic-resistant bacteria and particularly beta-lactamase-producing bacterial isolates are currently a major public health concern. The treatment of infections caused by such bacteria must be effective and promptly administered. For this phenotypical tests are a very useful tool, specifically in clinical laboratories, due to its functionality and low price to determine bacterial and fungal antibiotic resistance.
However studies comparing the efficiency and specificity of such tests are lacking.
For these reasons, the main objectives of this study were:
1) To evaluate the prevalence of AmpC-producers and ESBL-producers in “Centro Hospitalar de Coimbra, E.P.E.”;
2) To compare and validate several phenotypic methods to detect ESBL-producers and AmpC-producers;
3) To confirm the results of phenotypic methods by using genotypic methods;
4) To recommend a panel of phenotypical tests optimized and validated during this study to be applied in the laboratory routine.
27
29 3.1 Central Hospital characterization
All the isolates used in this study were collected from patients of “Centro Hospitalar de Coimbra, E.P.E.” (central Portugal) from September 2011 until October 2011.
The “Centro Hospitalar de Coimbra, E.P.E”. comprises the General Hospital (HG), the Pediatric Hospital of Coimbra (HP) and the Maternity Hospital of Bissaya Barreto, and it serves about 1,6 millions of people, from newborns to elderly people. This central hospital spans a very large area of the central region of Portugal, including Coimbra, Alvaiázere, Ansião, Castanheira de Pêra, Condeixa-a-Nova, Figueiró dos Vinhos, Montemor-o-Velho, Soure, Pedrógão Grande and Penela. It also serves Figueira da Foz, Leiria and Pombal.
In “Centro Hospitalar de Coimbra, E.P.E.” there is only one Laboratory of Microbiology, which is located in the General Hospital. This Laboratory receives around 35.000 biological samples per year, which are processed to detect potential pathogens and determine its resistance antibiotic profiles. The biological samples received are from the emergency room, the day care hospital, the outpatient and the internment, in the medical specialties of intensive care, cardiology, orthopedics and traumatology, neurology and neurosurgery, medicine, infectious diseases, pneumology, nefrology and urology, gastroenterology, ophthalmology, otorrino, gynecology and obstetrics, neonatology, hematological and oncological diseases.
After receiving the samples, biological products are processed to search for aerobic and anaerobic bacteria, yeasts and filamentous fungi, and mycobacteria. According to the origin of the sample and the type of analysis, the response time can vary from 3 to 50 days.
3.2 Bacterial Strains
During the timeframe of this study, 498 Enterobacteriaceae isolates belonging to the following species, E. coli, K. pneumoniae and K. oxytoca, were collected. The biological samples were collected from different cohorts: emergency room (ER) of HG and HP, day care hospital of HG, outpatient of HG and HP and internment of HG and HP, and sent to the Laboratory of Microbiology to its correct processing.
Urine samples were obtained from midstream and inoculated in CLED agar (cystine lactose electrolyte deficient medium) (BioMérieux, France). Only from cultures with a bacteriuria ≥ 1x104
CFU the pathogenic organism was isolated to CLED. All the other samples (sputum, tracheal aspirate, blood culture and abscess pus), collected according to the respective aseptic procedures, were inoculated in Columbia agar (COS) containing 5% of sheep red blood cells (BioMérieux, France). The isolation of the Enterobacteriaceae causing the infection was performed in the same medium. All the cultures were incubated for 16-24 hours at 37ºC in microaerophilic or aerophilic atmosphere, if it was a first culture or an isolation culture, respectively.
The identification and resistance antibiotic profile of all the isolates was obtained in the WalkAway System (Siemens, USA), using panels that combines biochemical tests for the identification and tests with several antibiotics to measure the bacterial growth in the presence of those antibiotics, to determine the resistance antibiotic profile of the microorganism. In the panels are included tests to detect ESBLs, which are done routinely. Among the 498 isolates studied, no isolate were identified as AmpC-producers, while 50 isolates were identified as ESBL-producers and included in this study: 25 E. coli, 24 K.
pneumoniae and 1 K. oxytoca.
3.3 Phenotypical tests to detect the production of beta-lactamases
3.3.1 Disc-based tests
The disc test used was MASTDISCSTM ID AmpC and Extended Spectrum Beta-Lactamase (ESBL) Detection Discs (MAST, UK).
This test is based in the bacterial behavior in the presence of cefpodoxime 10 µg alone (disc A), cefpodoxime 10 µg plus an ESBL inhibitor (disc B), cefpodoxime 10 µg plus an AmpC inhibitor (disc C) and cefpodoxime 10 µg plus an ESBL inhibitor and an AmpC inhibitor (disc D). Comparing the zone of inhibition of the cefpodoxime disc (A) to the zones of inhibition of each disc with cefpodoxime and an inhibitor (B, C and D), it can be determined if the organism tested has ESBL or AmpC activity or none of those.
31 The technique was executed according to the manufacturer’s specifications:
- Using a pure, fresh culture of the organism, prepare a suspension with 0.5 McFarland; - Using a sterile swab spread the suspension uniformly across the Mueller Hinton Agar plate, in 3 directions;
- place one of each type of MAST ID AmpC and ESBL Detection Set discs onto the inoculated medium, ensuring sufficient space between the discs to allow formation of clearly defined zones of inhibition;
- Incubate at 35-37ºC for 18 to 24 hours;
- Measure and record in “mm” the diameter of any zones of inhibition that are observed. Discs showing no zone of inhibition should be recorded as 6mm;
- To interpret the results, compare the zone of inhibition of the Cefpodoxime disc (A) to the zones of inhibition of each of the Cefpodoxime plus inhibitor discs (B, C and D).
a) if the results obtained by subtracting discs A from B and discs C from D are ≥ 5 mm and also subtracting discs B from D and the discs A and C are < 5 mm, then is considered that the organism only produces ESBL beta-lactamases;
b) if the results obtained by subtracting discs A from B and discs C from D are < 5 mm and also subtracting discs B from D and the discs A and C are ≥ 5 mm, then is considered that the organism only produces AmpC beta-lactamases;
c) if the results obtained by subtracting discs C from D are ≥ 5 mm but subtracting discs A from B < 5 mm, then is considered that the organism produce ESBL and AmpC beta-lactamases.
3.3.2 Strip-based tests
Cica-Beta-Test strip (MAST, UK) is a rapid screening test that allows detecting beta-lactamases, but not identifying a specific type of beta-lactamase. This test takes advantages of specific characteristics of certain antibiotics, such as the chromogenic capacity of cephalosporins.
The HMRZ-86, a chromogenic cephalosporin has a carboxypropyloxyimino group as a protective group for its beta-lactam ring, which prevents cleavage by beta-lactamases (penicillinase, cephalosporinase). However, extended-spectrum beta-lactamases (ESBLs
and metallo-beta-lactamases) are able to hydrolyze the beta-lactam ring in HMRZ-86, resulting in a colour change from yellow to red/pink.
Although, the Cica-Beta-Test can support in the guidance of an early therapeutic response, it must be used only in combination with susceptibility data.
The technique was executed according to the manufacturer’s specifications:
- Dispense one drop (approximately 0.02 mL) of Cica-Beta-Test substrate onto the filter pad of the Cica-Beta-Test strip, immediately before testing;
- Using a loop, pick several identical colonies from a pure, fresh culture. The colonies must be tested immediately to avoid misleading results;
- Spread the colonies on the filter pad of the Cica-BetaTest strip;
- Observe the test strip after 2 to 15 minutes at room temperature. Do not read the results after 15 minutes.
3.3.3 Etest (AB BioMérieux, Sweden)
This technique is based in the same principle of the disc tests. However, differs from the former in that the antibiotics are impregnated in a cellulose strip with a concentration gradient of a specific antibiotic, the Etest strip. To determine the presence of beta-lactamases, the strips are divided in two regions, in one top with only the antibiotic impregnated, in a crescent concentration gradient from the center to the top of the strip, and in the other top the same antibiotic is impregnated in association to a beta-lactamase inhibitor.
As in the disc test, it is possible to identify the type of beta-lactamase produced, according to the Etest (AB BioMérieux, Sweden) used.
The Etest strips used to detect the production of ESBLs were:
- CT/CTL (strips containing cefotaxime and cefotaxime + 4 µg/ml clavulanic acid); - TZ/TZL (strips containing ceftazidime and ceftazidime + 4 µg/ml clavulanic acid); - PM/PML (strips containing cefepime and cefepime + 4 µg/ml clavulanic acid). The Etest strips used to detect the production of AmpC beta-lactamases were: - CN/CNI (strips containing cefotetan and cefotetan + 4 µg/ml cloxacillin). The technique was executed according to the manufacturer’s specifications:
33 - Using a sterile swab, spread the suspension uniformly across the Mueller Hinton Agar plate, in 3 directions;
- place the Etest strip onto the inoculated medium, ensuring that the scale of the strip is visible;
- Incubate at 35-37ºC for 18 to 24 hours;
- Read MIC values and note “phantom” zones or deformation of inhibition ellipses.
3.4 Semi-automatic tests
The WalkAway System (Siemens, USA), shown in figure 6, is a semi-automatic system that allows to identify Gram-positive and Gram-negative bacteria and to determine their antibiotic susceptibility profile. For this are used MicroScan panels (Siemens, USA), shown in figure 7, which include a range of substrates for the identification of the isolate and substrates to determine the corresponding antibiotic susceptibility profile. Different panels are used depending if the bacteria is Gram-positive or Gram-negative and also, in the case of Gram-negative bacilli, according to the nature of the biological product from which the bacteria was isolated.
In Dried Gram Negative MIC/Combo Panels and Dried Gram Negative Breakpoint Combo Panels, modified conventional and chromogenic tests are used for the identification of fermentative and non-fermentative Gram-negative bacilli. The identification is based on the detection of pH changes, the substrate utilization (colorimetry) and the growth in the presence of antimicrobial agents after 16-42 hours of incubation in WalkAway at 35ºC (MacFaddin JF, 2000).
After inoculation and rehydration of the antibiotic from the panel with a standardized suspension of bacteria and incubation in WalkAway at 35ºC for a minimum of 16 hours, the minimum inhibitory concentration (MIC) is determined for the organism tested by the determination of the lowest antimicrobial concentration showing inhibition of growth (CLSI, 2007). The panels containing ceftazidime, aztreonam, cefotaxime or ceftriaxone at 1 µg/ml or cefpodoxime at 1 or 4 µg/ml (depending on panel type), were used to screen for
Figure 6 – WalkAway System equipment (Siemens, USA).
Figure 7 – Example of a MicroScan panel NBC 43 for identification and antibiotic profile for Gram-negative bacteria isolated from other samples than urine (Siemens, USA).
35 3.5 Genotypic tests
3.5.1 DNA extraction
DNA extraction was performed, in all the 50 isolates identified as ESBL-producers, by collecting 1 or 2 colonies of a pure culture with a sterilized plastic stick and suspending the colonies in 100 μl of distilled and sterilized water in a sterile microtube. The suspensions were stored at room temperature for 24 hours. Before using in the PCR, a short-spin was done to the suspensions and the supernatant was used in PCR reactions. The suspensions may be stored in the refrigerator at 2-8ºC for one week.
3.5.2 Molecular typing of bacterial isolates
PCR-based molecular typing methods (specifically BOX-PCR and ERIC-PCR) were applied as described before (Versalovic et al., 1991) to evaluate the molecular diversity of the isolated bacteria. PCR was performed using DNA extracts from pure cultures. Twenty-five µl reaction mixtures contained: 2.5 µl PCR buffer 10X, 2.5 µl MgCl2 25 mM, 1.5 µl primers 0.3 pmol/L (forward + reverse), 3 µl DNTPs 10 mM, 0.5 µl Taq polymerase 5U/µl, 14 µl distilled and deionised water and 1 µl bacterial DNA. Primers used are presented in table 4. The PCR program for BOX-PCR was as follows: initial denaturation (95ºC for 7 min); 30 cycles of denaturation (94ºC for 1 min), annealing (53ºC for 1 min as indicated in table 4), and extension (65ºC for 8 min); and a final extension (65ºC for 16 min). The PCR program for ERIC-PCR was: initial denaturation (94ºC for 3 min); 30 cycles of denaturation (94ºC for 30 sec), annealing (52ºC for 3 min as indicated in table 4), and extension (68ºC for 8 min); and a final extension (68ºC for 8 min).
3.5.3 Detection of beta-lactamases-encoding genes
A set of selected isolates was screened by PCR for the presence of genes blaSHV,
