Susceptibilities to carbapenems and presence of cphA gene on food-borne Aeromonas

Abstracts

The purpose of this study was to determine the susceptibilities of food-borne Aeromonas to carbapenems, as well as to investigate the presence of a metallo carbapenemase-encoding gene, named cphA. Minimum Inhibitory Concentration (MIC) was determined following NCCLS standards. All the tested microorganisms were susceptible to imipenem, meropenem and biapenem. However, a strong inoculum size effect on carbapenem MICs was observed for most of the strains. Six strains, out of seven, showed the presence of metallo--beta-lactamases but cphA gene was detected in only two strains of A. veronii bv. sobria.

metallocarbapenemase; cphA gene; food borne Aeromonas

O objetivo deste estudo foi determinar a suscetibilidade de aeromonas de origem alimentar a carbapenems bem como investigar a presença de um gene codificante de metalocarbapenemase, denominado "cph A". A suscetibilidade in vitro foi determinada pelo metodo de diluição em agar. Todas as cepas foram suscetíveis a Imipenem, Meropenem e Biapenem. Porém foi observado um forte efeito de tamanho do inóculo sobre as CIM das carbapenems na maioria das cepas. A detecção de metalo-beta-lactamase foi realizada pelo metodo lodometrico. Seis cepas das sete testadas demostraron a presença da enzima. A presença do gene cphA foi determinada por PCR e foi detectada em duas cepas de A veronii bv. sobria.

FOOD SCIENCE AND TECHNOLOGY

Susceptibilities to carbapenems and presence of cphA gene on food-borne Aeromonas

Bibiana María Martín Talavera^I; Fernando Olivio Benassi^I; Martha Helena von SpechtI, ^*; Marina Inés Quiroga^I; Myriam Alicia García^I; Amanda Beatríz Pucciarelli^I; Emilce Zubreski^I; Margarita Esther Laczeski^I; Gabriel Gutkind^II

^ICentro de Investigación y Desarrollo Tecnológico - Bidet; Facultad de Ciencias Exactas, Químicas y Naturales; Universidad Nacional de Misiones; Félix de Azara, 1552; 3300 Posadas; Misiones - Argentina

^IICátedra de Microbiología; Facultad de Farmacia y Bioquímica; Universidad de Buenos Aires; Buenos Aires - Argentina

ABSTRACT

The purpose of this study was to determine the susceptibilities of food-borne Aeromonas to carbapenems, as well as to investigate the presence of a metallo carbapenemase-encoding gene, named cphA. Minimum Inhibitory Concentration (MIC) was determined following NCCLS standards. All the tested microorganisms were susceptible to imipenem, meropenem and biapenem. However, a strong inoculum size effect on carbapenem MICs was observed for most of the strains. Six strains, out of seven, showed the presence of metallo--b–lactamases but cphA gene was detected in only two strains of A. veronii bv. sobria.

Key words: metallocarbapenemase, cphA gene, food borne Aeromonas.

RESUMO

O objetivo deste estudo foi determinar a suscetibilidade de aeromonas de origem alimentar a carbapenems bem como investigar a presença de um gene codificante de metalocarbapenemase, denominado "cph A". A suscetibilidade in vitro foi determinada pelo metodo de diluição em agar. Todas as cepas foram suscetíveis a Imipenem, Meropenem e Biapenem. Porém foi observado um forte efeito de tamanho do inóculo sobre as CIM das carbapenems na maioria das cepas. A detecção de metalo-b-lactamase foi realizada pelo metodo lodometrico. Seis cepas das sete testadas demostraron a presença da enzima. A presença do gene cphA foi determinada por PCR e foi detectada em duas cepas de A veronii bv. sobria.

INTRODUCTION

Evolution of bacterial resistance to antibiotics in humans, animals and the environment is the result of the interaction between the exposure to antibiotics, selection of microorganisms carrying primordial genes of resistance, and transmission of resistance genes between bacteria. Selective effects occur in selective compartments, where particular antibiotic concentrations result in a differential growth rate of resistant bacterial variants (Baquero et al., 1998) This may happen even at very low antibiotic concentrations able to select low-level-resistant bacteria. Analysis of selective environment-related antibiotic-host-bacteria interactions is essential to understand the biology of antibiotic resistance. (Baquero et al., 1998)

To anticipate emergence of resistance, it is necessary to better understand the genetics and biochemistry of resistance mechanisms and to develop methodologies to foresee their evolution at the individual or population level (Baquero et al., 1998). Most retrospective and prospective studies show that after the introduction of an antibiotic, the level of resistance increases both among pathogenic bacteria and in commensal bacteria (van den Bogaard and Stobberingh, 2000). Moreover, commensal bacteria constitute a reservoir of resistance genes for (potentially) pathogenic bacteria. Their level of resistance is considered to be a good indicator for selection pressure by antibiotic use and for resistance problems to be expected in pathogens.

Aeromonas strains have been found to be able to produce up to three different b-lactamases including a group 1 (according to the Bush-Jacoby-Medeiros classification) molecular class C cephalosporinase, a group 2d molecular class D penicillinase and a group 3 molecular class B metallo b-lactamases (Hayes et al., 1994; Walsh et al., 1995). Among the different types of b-lactamases found in aeromonads, the molecular class B enzymes are particularly interesting from the clinical standpoint because of their ability to hydrolyze carbapenem compounds (which are broad-spectrum antibiotics not hydrolyzed by most other b-lactamases) and contribute to resistance against these antimicrobial agents (Massida et al., 1991; Morita et al., 1994; Rossolini et al., 1995; Sanders et al., 1989). Metallo-b-lactamases (class B of the molecular classification of Ambler or group 3 according to the functional classification of Buch et all) constitute a very heterogeneus family. (Mercuri et al., 2001).

On the basis of structural analysis, these enzymes cluster into three different groups: subclass B1 contains most known zink-b-lactamases for example BcII from Bacillus cereus 569H (Hussain et al., 1985) and the plasmid-encoded enzyme IMP-1 found in some isolates of Pseudomonas aeruginos, Serratia marcescens and other Gram negative bacteria (Laraki et al., 1999; Osano et al., 1994), subclass B2 includess the Aeromonas enzymes (CphA, ImiS, and CphA2; Mercuri et al., 2001) and subclass B3 contains for example the tetrameric L1 enzyme produced by Stenotrophomonas maltophilia (Sanchagrin et al., 1998).

The enzymes from Aeromonas species are known as "true carbapenemases". These enzymes have a high specificity for hydrolyzing carbapenems and cannot be detected with nitrocefin. (Rossolini et al., 1996) In a survey performed on reference Aeromonas strains of several different species, it was observed that production of carbapenemase activity was restricted to strains of some species, being not detectable in others and that in all cases this activity was inhibited by EDTA (Rossolini et al., 1995) An A. hydrophila metallo carbapenemase-encoding gene, named cphA, has been cloned from a clinical isolate (Massida et al., 1991) Experiments showed that cphA-specific probes recognize A. hydrophila, A. veronii bv. sobria, A. veronii bv veronii, A jandaei, A. salmonicida (both subsp. salmonicida and subsp. achromogenes) but not A. caviae (with a few exceptions), A. trota, or A. schubertii strains (Rossolini et al., 1995; Rossolini et al., 1996)

Similar to other chromosomally encoded b-lactamases types, the production of the class B enzyme is normally regulated in Aeromonas strains: the enzyme is produced at negligible levels in the absence of suitable inducers, while production increases several hundredfold in the presence of a suitable b-lactam inducer such as penicillin or imipenem (Hayes et al., 1994; Massida et al., 1991; Segatore et al., 1993; Walsh et al., 1995). Aeromonas spp. are indigenous to acquatic environments (Warren et al. 2004), and have become increasingly implicated as causative agents of food borne associated human diseases such as gastroenteritis (Abeyta et al., 1986; Kirov et al., 1990). Accute diarrheal outbreaks in long-term care settings were reported (Bloom et al., 1990).

The aim of this work was to study the susceptibility patterns of carbapenems on Aeromonas strains, isolated from chicken carcasses, and to detect cphA gene presence on Aeromonas sp. producing a carbapenemase activity.

MATERIALS AND METHODS

Bacterial strains

Seven Aeromonas strains isolated from chicken carcasses were used in this study. These were A veronii biov. sobria (3) and A caviae (4) (Benassi et al., 2001).

In vitro susceptibility tests

MIC's of selected antibiotics were tested by agar dilution method (National Committee for Clinical Laboratory Standards: 1999; NCCLS 2003) with Mueller Hinton Agar (Bickar, France), with inocula of 10⁵ (normal) and 10^7-8 (high) CFU/per spot on the plate using a Steers replicator. MIC's were determined the following compounds: Imipenem (IMP- Merck Sharp & Dohme Research Laboratories - Rahway, N. Y., USA), Meropenem (MER - Zeneca Pharma - Italy), Biapenem (BIA- Cyenamid -Italy). These were obtained as powders with known potency.

b -lactamase production

Microorganisms were grown overnight in 50 ml of Brain Heart Infusion (BHI- Britania, Argentina), contained in 250 m l flasks at 37°C and 200 rpm. Cultures were collected 2-h after induction with 100 µg/ml anhydrous ampicillin, centrifuged at 10,000 x g and resuspended in 5 ml of 30 mM sodium phosphate buffer (pH 7.2). The cells were disrupted by vortexing with sand (HCl washed) (particles 0.10 - 0.15 mm, 1:1, w/v sand to cells ratio, 10 x 1 min each, 0 °C). Crude cells extracts were centrifuged (15,000 x g) to remove bacterial debris and were stored at -20 °C until used.

Determination of b-lactamase activity

Detection of b-lactamase was performed by an iodometric technique with 1000 µg/ml IMP as the substrate (Quinteros et al., 1990).

Inhibition experiments

The susceptibility of b-lactamases to inhibition by EDTA (10 mM final concentration) was determined by the iodometric method after incubation of the crude extracts (20 µl) for twenty minutes at 25 ºC in the presence of 10 µM EDTA. IMP (1000 µg/ml) was used as the substrate. In all cases, a control assay without EDTA was run in parallel.

PCR Analysis

The PCR analysis to detect the presence of cphA gene was performed as described by Massida et al. Cromosomal DNA was extracted from the carbapenemase producers strains. The set of primers used was ANY-SSD/F (5' GCT TAG AGC TCC TAA GGA GCA AGA TGA AAG GTT GG 3' and 5' GCA TAG GTA CCT TAT GAC TGG GGT GCG GCC TTG 3') (Massida et al.,1991).

RESULTS

Table 1 shows MIC for IMP, MER and BIA of the strains studied. All the strains were susceptible to IMP: CIM₉₀ = 0.5 µg/ml, MER: CIM₉₀ = 0.125 µg/ml, and BIA: CIM₉₀ = 0.125 µg/ml. When a large inoculum size was used, an impact was observed: 85.7% for imipenem, 71.5% for meropenem and 100% for biapenem. (Table 1). Table 2 shows the results of the metallo b-lactamase experimentsand the presence of the cphA gene.Figure 1 presents the results of PCR amplification with primers ANY-SSD/F of the cphA gene.

DISCUSSION

It has been reported that metallo-b-lactamase mediated resistance phenotype could not readily recognized in conventional in vitro susceptibility testing (Bakken et al., 1988; Hayes et al., 1994; Massida et al., 1991; Rossolini et al 1995). This appeared to be the case for carbapenems antibiotics and carbapenemase-producing strains in the present study also. Results showed a strong inoculum size effect on carbapenem MICs, with few A. caviae as exceptions. This effect was also observed Rossolini et al. (1996) who related it with carbapenemase producing Aeromonas strains. In fact, using larger inocula (10⁸ CFU/ml), carbapenems MICs for these strains become usually higher than the breakpoint for susceptibility, while carbapenems MICs of Aeromonas strains that do not express a carbapenemase activity (such as the majority of A. caviae strains) remain always below the breakpoint for susceptibility. This seems to be the situation largely found in most of contaminated foods related with foodborne diseases.

EDTA inhibition of carbapenems activity suggested their metallo enzimes nature.Rossolini et al (1995) found that production of metallo carbapenemase activity was restricted to strains of some species, being not detectable in others, and that in all the cases this activity was inhibited by EDTA. However, a minority of A. veronii bv. sobria strains was found to be unable to produce the enzyme, while a minority of A. caviae strains expressed similar activity.

Although most of the phenotypes expressed by the strains studied were of carbapenemase activity, cphA gene aleles was detected in two strains of A. veronii bv sobria. However, it appeared more likely to consider that other carbapenemases (still to be characterized), might be present in strain 79, different enough to cphA gene as not to react with the specific primers. The lack of its presence in A. caviae was in accordance with other research findings (Rossolini et al. 1995, 1996). Furthermore, it would be always advisable to perform susceptibility testing, also with large inocula, or looking for carbapenemase production. The possibility of horizontal transfer of cphA alleles to strains of species that normally do not carry it, must be considered. Most known metallo-b-lactamases are encoded by chromosomal genes of some bacterial species that are primarily members of the environmental microbiota, such as Aeromonas spp., whereas some as yet unknown environmental species are the most likely sources of the mobile metallo-b-lactamases determinants that recently appeared among gramnegative patogens. (Rosolini et al., 2001)

The emergence of resistant bacteria and resistance genes following the use of antimicrobial agents is relatively well documented and it seems evident that all antimicrobial agents would select for resistance (Aarestrup 1999). The occurrence of a transmitted food disease due to metallo-b-lactamases producers microorganisms would limit the efficiency of a carbapenem-based chemotherapy in the treatment of human infections.

Received: September 23, 2004;

Revised: March 02, 2005;

Accepted: March 14, 2006.

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*

Author for correspondence

Publication Dates

• Publication in this collection
  30 Aug 2006
• Date of issue
  July 2006

History

• Accepted
  14 Mar 2006
• Reviewed
  02 Mar 2005
• Received
  23 Sept 2004