Metallo-beta-lactamase producer Pseudomonas aeruginosa: an opportunistic pathogen in lungs


Chapter 10

Metallo-beta-lactamase producer Pseudomonas aeruginosa: an opportunistic pathogen in lungs



S.U. Picoli*

A.L.S. Gonçalves**
*    Universidade Feevale, Novo Hamburgo, Rio Grande do Sul, Brazil
**    MSc at Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil


Abstract


Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that is mostly involved with hospital infection worldwide. It may display different antimicrobial resistance mechanisms, with emphasis on the enzymatic mechanisms; for instance, the metallo-beta-lactamase production. These enzymes inactivate virtually all beta-lactam antibiotics, except monobactams. Infections caused by metallo-beta-lactamase producing Pseudomonas aeruginosa are a clinical and epidemiological challenge because metallo-enzymes genes are linked to plasmids, highly mobile genetic elements, which enable their rapid spread. High morbi-mortality rates are described, particularly in patients in Intensive Care Units with respiratory infections associated with mechanic ventilation. Treatment of acute infections, such as pneumonia, and chronic infections, like cystic fibrosis, is a constant clinical challenge. For the clinical laboratory, detection of metallo-beta-lactamase producing isolates also represents a frequent challenge because there is no standardization of described methods and often, necessary resources are not available for rapid diagnosis.



Keywords


Pseudomonas aeruginosa

metallo-beta-lactamase

carbapenemase

multidrug resistance

lung infection


1. Introduction


Pseudomonas aeruginosa is an opportunistic human pathogen that is frequently isolated in hospital infections, especially in nosocomial pneumonia.1 Species of this genus are characterized by their inability to ferment sugars, although most of the strains oxidatively degrade them. However, they are nutritionally versatile bacteria that are able to use a wide variety of substrates, whose optimum growth temperature varies between 30 and 37°C, although they multiply in lower temperatures.2 Furthermore, P. aeruginosa has phenotypic and biochemical characteristics that allow its easy laboratorial identification; for instance, the production of water-soluble pigments named pyocyanin (white to blue-green fluorescence under ultraviolet light) and pyoverdine, in addition to producing a characteristic grape-like odor.3

Given that it is considered to be an opportunistic pathogen, it is generally not a high risk for healthy individuals, although it has been reported as a recurrent community-acquired pneumonia in a young patient who had no risk factors.4 Adversely, due to the wide distribution of these bacteria in the environment, it may be problematic, especially in hospitals, since it remains on artificial respiratory systems used in Intensive Care Unit (ICU) patients, as well as in aqueous solutions.2 Besides these features, P. aeruginosa is capable of producing several virulence factors, which are strongly related to this bacteria potential to cause disease.5 This ability is mediated by the presence of genes that express different virulence factors, including exoS (exoenzyme S), exoU (exoenzyme U), toxA (exotoxin A), LasB (elastase LasB), nan1 (neuraminidase), plcN (nonhemolytic phospholipase C), plcH (hemolytic phospholipase C), pilB (type IV fimbrial biogenesis protein pilB), algD (alginato), among others. The latter gene is responsible for the production of a mucopolysaccharide capsule that gives greater adherence of the bacteria to the respiratory epithelial cells, forming biofilms, which hamper the action of antimicrobial agents and immune system. Regarding the pathogenic potential of P. aeruginosa, another important characteristic is the simultaneous presence of several of this described virulence genes and enzymes, such as, the metallo-beta-lactamases (MBL). This association has a significant correlation between the virulence factors and antibiotic resistance patterns in P. aeruginosa multidrug resistant (MDR-PA) isolates.6 Indeed, plasmids are one of the most important virulence mechanisms in multidrug resistant bacteria. They transport antibiotic resistance determinants contained in transposons (mobile genetic elements), which results in constant modifications to DNA.7

Nevertheless, patients hospitalized in ICUs with severe underlying diseases, previous antimicrobial treatment, undergoing invasive procedures, as well as extended periods of hospitalization, are of particular risk of acquiring nosocomial infections by P. aeruginosa. These conditions contribute to the association of the bacteria with high rates of morbidity and mortality in critically ill patients in ICUs, particularly in those with mechanical ventilator associated pneumonia.8 In addition, exposure to several antimicrobial agents in the hospital environment may create conditions for resistance selection among the host microbiota or for pathogen transmission. Consequently, transfer of patients between admission units in the hospital may provide the introduction of new and often highly resistant clones into the ICU.9

2. P. aeruginosa resistant to beta-lactam antibiotics


Usually, both intrinsic and acquired wide resistance to numerous antibiotics used in medical practice contribute to P. aeruginosa’s pathogenicity, since bacteria from this genus may demonstrate high rates of resistance to most of the antimicrobial agents normally used in hospital environment. Therapeutic options include aminoglycosides, fluoroquinolones, broad-spectrum penicillins, monobactams, ceftazidime, fourth generation cephalosporins and carbapenems. One of the recommendations for antibiotic therapy in treatment of infections caused by P. aeruginosa is the use of association including a beta-lactam and another antibiotic, usually an aminoglycoside or quinolone, being the beta-lactams the basis of the chosen association.10

However, P. aeruginosa resistance to beta-lactam antibiotics may occur due to enzymatic mechanisms, for instance, the beta-lactamases production via the activation of outer membrane impermeability systems, which are related to porins11 and also via antimicrobial agents expulsion of the bacteria cell, called the efflux pump.10,11 Eventually, more than one mechanism may be present, resulting in a wide resistant pattern to all beta-lactams, being the beta-lactamases production the most studied mechanism over the last few decades.

MBL is an enzyme classified in the 3a group of Bush–Jacoby, in which all the biggest families of MBLs codified by plasmids are included, such as, the IMP (IMiPenemase) and the VIM (Verona Integron-encoded Metallo-beta-lactamase). They have high hydrolytic potential on penicillins, cephalosporins, carbapenems, but not on monobactams. Due to zinc metal (Zn2+) present on the enzyme active site, it can be inhibit by metal chelating agents, such as, EDTA (ethylenediaminetetraacetic acid), but not by beta-lactamases inhibitors clinical available, like clavulanic acid or tazobactam.12

The emergence of MBL producing microorganisms has great clinical importance, as they show resistance to many antibiotics used, with sensitivity only to colistin and polymyxin B, limiting treatment options. Therefore, an appropriate screening system should be established, especially for all P. aeruginosa imipenem-resistant, enabling early detection of carbapenemase strains, and hence, preventing the spread of these multidrug resistant strains.13

MBLs are categorized in types according to its amino acid’s sequence. More than 10 MBL variants were described and most of them have been already found in P. aeruginosa: IMP (IMiPenemase), VIM [Verona Integron-encoded Metallo-beta-lactamase, SPM (São Paulo Metallo-beta-lactamase)], GIM (Germany IMipenemase) NDM (New Delhi Metallo-beta-lactamase), FIM (Florence IMipenemase).1418 The distribution of these enzymes is worldwide and they have been described in different countries of America, Europe, Asia, Africa and Oceania, with mean of variation between 10% and 50%.19 However, its constant local monitoring is recommended, since epidemiology of these carbapenemase not only varies among different countries but also within the same country.

Thus, this monitoring becomes more relevant knowing that metallo-beta-lactamase producing P. aeruginosa (MBL-PA) isolates are associated to infection’s rapid progression and evolution to death. In addition, MBL-PA are more resistant than non-MBL-PA strains and high clonal dissemination of MBL-PA strains suggest the cross transmission as an important mechanism of spreading.20 Considering that resistance rates to polymyxin B tend to increase after generalized use of this drug, strict infection control measures should be urgently adopted, otherwise we may be facing untreatable P. aeruginosa nosocomial infections.21

Not all patients on mechanical ventilation develop lung tissue infection, since it depends on other factors such as host defenses and the P. aeruginosa virulence colonizing the lower respiratory tract. Nevertheless, the ventilator associated pneumonia (VAP) is an important respiratory infection seen in ICU patients and it occurs in 7–20% of those who were mechanically ventilated for periods greater than or equal to 48 h.22 VAP is initiated by an inflammation of the lung parenchyma caused by aspirated microorganisms after mechanical ventilation and results in increased hospitalization time, higher treatment cost and higher mortality.23 Additionally, when VAP is caused by P. aeruginosa the infection becomes invasive with rapid progression. It is characterized by acute leukocytosis and fever, which requires increased ventilator support besides raising significantly the mortality rates.24,25 Production of MBL might be associated to higher mortality due to inadequacy of antimicrobial therapy in infections caused by MBL-PA, suggesting that institutions with high prevalence of MBL should review their therapeutic approaches.24

Commonly, nosocomial P. aeruginosa isolates obtained from respiratory material as bronchoalveolar lavage or endotracheal aspirate show antimicrobial resistance mediated mainly by MBLs. Additionally, biofilm formation on endotracheal tube, with subsequent embolization to distal respiratory tract, might be important in the pathogenesis of VAP.26,27

Microbiological criteria for VAP are quantitative and they vary according to the type of clinical material under study, where in findings of ≥106 colony forming units (CFU)/mL in endotracheal aspirate and ≥104 CFU/mL in bronchoalveolar lavage are considered.25 Also, according to the American Toracic Society, all patients with suspected VAP should have blood cultures collected and a positive result can indicate the presence of pneumonia or extrapulmonary infection.28 However, hospital-acquired pneumonia index seen in nonventilated patients is also high (>70%) and the etiological agents are similar to those found in patients with VAP, including P. aeruginosa multidrug resistant (MDR-PA).8,29 Higher survival rate is expected in these patients when polymyxin is used by inhalation in addition to antimicrobial therapy,30 noting that a study demonstrated that only this antibiotic showed overall coverage equal to or superior than 90% in patients with VAP.31

The result of multivariate analysis, conducted by Zavascki et al.,32 showed that recent use of a beta-lactam antibiotic (OR 3.21; 95% CI 1.74–5.93) or a quinolone (OR 3:50; 95% CI 1:46–8:37) was a significant risk factor for infection caused by MBL-PA, even though patient to patient transmission has a major role in the dissemination of isolates. Lung was shown to be the most frequent nosocomial infection site in the patients who were evaluated (50.3%).

Regarding the chronic infections that affect lungs, cystic fibrosis (CF) is presented as the most common among Caucasians. This disease is characterized by airway obstruction due to the production of thick and viscous secretions, which increases the individual’s susceptibility to pulmonary infections, being P. aeruginosa one of the main bacteria involved in these infections.33,34

Conversely, it is not entirely clear why infections in CF are chronic, noninvasive and highly resistant to eradication. Production of biofilms has been regarded as the main mechanism, but a study showed that P. aeruginosa might grow in bacterial aggregates, which are greatly resistant to host defenses and to antibiotics independently of the bacterial biofilm production.35 Moreover, MBLs genes (IMP and SPM) were found concomitantly to biofilm production in P. aeruginosa CF isolates, suggesting that overlapping antibiotic resistance mechanisms represents a therapeutic challenge even greater than the one faced in isolates that only produce biofilms.36

However, the majority of CF patients acquire chronic P. aeruginosa infections in early life, which is responsible for much of the morbidity and mortality in individuals with the disease. Furthermore, hypermutable strains of P. aeruginosa were isolated from an elevated percentage of CF patients. P. aeruginosa follows a characteristic evolution pattern defined by selection of hypermutable strains and increase of antibiotic resistance as a consequence of long term infection in CF-affected lung. Besides affecting CF patients, P. aeruginosa is also recognized as a relevant pathogen in chronic obstructive pulmonary disease (COPD). These individual often have acute exacerbations, which cause great impact on these patients’ quality of life. These exacerbations are the main cause of mortality among patients affected by this disease.37

3. Detection tests of MBLs in P. aeruginosa


P. aeruginosa isolates with phenotype of nonsusceptibility to antipseudomonal broad-spectrum antibiotics such as carbapenems (imipenem and meropenem) may be carriers of some MBL gene. In this context, it is essential that the microbiology laboratory promptly detect the metallo-enzyme, enabling the appropriateness of antimicrobial therapy and contributing to making infection control measures.

After observing the carbapenem-resistance phenotype, either by agar diffusion test or by automation system, it is important to confirm the MBL presence. Different methods can be used, ranging from phenotypic tests that are easy to perform and inexpensive, to even the most sensitive and specific ones like the molecular tests (Polimerase Chain Reaction-PCR, DNA probes, cloning, sequencing)38 and MALDI-TOF MS (Matrix-Assisted Laser Desorption-Ionization Time-Of-Flight Mass Spectrometry).39 Unfortunately, the majority of these last resources are not available in most of the clinical laboratories.

The following tests may be produced in-house in clinical laboratories, and choice of any of them for routine investigation of MBL should consider the local MBLs epidemiology. Nevertheless, these tests are prone to error due to the great diversity of the enzymes types present in this group (MBLs in P. aeruginosa: VIM, IMP, SPM, NDM, GIM, and FIM).

Other factors that may contribute to disagreeing results in MBL detection are: trustworthiness of the inputs used, the concentration of enzyme inhibitor (EDTA) on the carbapenem disk and also, operational errors inherent to test preparation.40 One of the major problems faced on phenotypic MBL screening is the lack of standardization of a single test.

3.1. Combined disk (CD) test


Variants of this test (Table 10.1) have in common the inoculum preparation equivalent to 0.5 McFarland of the microorganism under test. The inoculum is seeded on Mueller Hinton agar surface (MHA); two disks of the same carbapenem are placed on the agar and enzyme inhibitor (EDTA) is added to one of them (Fig. 10.1). After suitable incubation, typically for 24 h at 35°C, the inhibition diameters difference between the carbapenem disk with EDTA and the carbapenem alone are measured, which leads to a positive or negative result for MBL.


Table 10.1


Phenotypic Tests for Metallo-Beta-Lactamase (MBL) Detection in Pseudomonas aeruginosa Isolates Resistant to Carbapenems


































































Song et al.41 Sheikh et al.40 Qu et al.42 Pitout et al.43
2 carbapanem disks IPMa 10 μg DORb 10 μg IPMa 10 μg MEMc 10 μg

EDTAe


aqueous solution

10 μL 10 μL 0.5 Md 10 μL 0.5 Md 930 μg
(30 mg/mL) (750 μg) (750 μg)

MBLf


presence

IPMa/EDTAe – IPMa DORb/EDTAe – DORb IPMa/EDTAe – IPMa MEMc/EDTAe – MEMc
≥5 mm ≥7 mm ≥6 mm ≥7 mm
Sensibility 100% 100% 100% 100%
Specificity 100% 64% 100% 97%

Types of MBLsf


detected

IMPg-6 IMPg IMPg-1 IMPg
IMPg-26 VIMh IMPg-9 VIMh
VIMh-2
VIMh-2



a  IPM: Imipenem.


b  DOR: Doripenem.


c  MEM: Meropenem.


d  M: Molar.


e  EDTA: Ethylenediaminetetraacetic acid.


f  MBL: Metallo-beta-lactamase.


g  IMP: Imipenemase.


h  VIM: Verona Integron-encoded Metallo-beta-lactamase.


image

Figure 10.1 Phenotypic Detection of Metallo-Beta-Lactamase in Pseudomonas aeruginosa by the Combined Disk Test (CD Test)Above/Upper: two meropenem disks (MEM 10 μg), plus 930 μg EDTA43 on the right disk; MEM/EDTA—MEM = 10.2 mm (MBL positive if ≥7 mm).
Bellow/Bottom: two imipenem disks (IPM 10 μg), plus 750 μg EDTA42 on the right disk; IPM/EDTA—IPM = 8.4 mm (MBL positive if ≥6 mm).

3.2. Carbapenem Inactivation Method


The Carbapenem Inactivation Method (CIM) is a new phenotypic test of low cost, which aims to detect the activity of different carbapenemases in Gram-negative bacilli, including MBLs of IMP and VIM types in P. aeruginosa.

The test consists in suspending a dense inoculum of the bacteria (a full 10 μL inoculation loop) in 400 μL of water. A meropenem (MEM) 10 μg disk is added in this suspension and it is incubated for at least 2 h at 35°C. The disk is removed from the suspension and it is placed on Mueller Hinton agar plate that was previously inoculated with Escherichia coli strain (ATCC 25922) adjusted to 0.5 McFarland. The plate is again incubated at 35°C for at least 6 h, although overnight incubation would be ideal. For interpretation, it is considered carbapenemase producing bacteria in the absence of any inhibition zone around the MEM disk because bacteria present in the suspension inactivates this antibiotic.

CIM results had high concordance (98.8%) when compared to the results of molecular tests (PCR) to detect specific genes involved in the resistance. Similarly, positive and negative predictive values were also high, determined at 96.3 and 99.4%, respectively.44

Alternatively, some quick tests for the detection of carbapenemase production may be performed. Among them is the Carba NP (homemade) that is based on the detection of beta-lactam ring hydrolysis of imipenem.45 This assay has been extensively validated for carbapenemase detection, both in Enterobacteriaceae and in Pseudomonas spp.4648 Reading is performed within 2 h, showing high sensitivity (94.4 %) and specificity (100%).47 In the commercial version of this test, named Rapidec Carba NP, the class B carbapenemase producing samples, the MBL, show a positive colorimetric response in less than 10 min.

3.3. Nonphenotypic tests


MALDI-TOF MS is one of the most promising and attractive carbapenemase detection techniques in clinical isolates. It has been introduced in the routines of different clinical laboratories because results can be obtained in shorter time when compared to other methods. Despite requiring a high initial investment, this technique allows early identification of a resistance mechanism, making it extremely useful for the initiation of antimicrobial therapy and it assists in controlling the transmission of this bacteria.

MALDI-TOF MS detects the activity of different carbapenemases with high sensitivity (95–99%), including the MBLs, via carbapenem hydrolysis. Briefly, the technique consists in preparing a very dense inoculum (equivalent to 3 McFarland) of the bacteria in a buffer and centrifuge. The pellet is suspended in a buffer containing the carbapenem molecule and it is incubated at 35°C for 4 h. Afterwards, it is centrifuged, a proper matrix (dihydroxybenzoic acid in 50% ethanol) is mixed to the supernatant, and measurement is performed by MALDI-TOF MS. Subsequently, spectra containing peaks representing the carbapenem molecule, its salts or its degradation products are analyzed.49

4. Conclusions and future perspectives


Respiratory infections associated with opportunistic bacteria such as metallo-beta-lactamase producing P. aeruginosa (MBL-PA) progresses rapidly, resulting in high morbi-mortality rates. The overall increase of this resistance has been reported in several countries and it is in evidence among the scientific community. Although several studies have been performed in an effort to establish a standard test for quick research of this powerful resistance mechanism in P. aeruginosa, it has not yet been possible to elect a single reliable test. This condition can be attributed to the diversity of circulating MBLs types (VIM, IMP, SPM, GIM, NDM, and FIM), reinforcing the need for local molecular epidemiological studies of these enzymes. It is presumed from prior knowledge of this data that it will be possible to establish a quick test, preferably with low cost, reliable, and feasible to be used in clinical laboratories covered by specific geographical location. However, it is evident that strict infection control measures, including restrictive antibiotic use policies in the hospital environment, isolation of patients with MBL-PA, especially ICU patients, should be applied whenever necessary, aiming to avoid the increase of nosocomial infections associated with MBL-PA.


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