I-Abstract

Antibiotic resistance is one of the major healthcare problems in the current time. Every day, healthcare is losing battles and pathogens are gaining new lands. Pseudomonas aeruginosa is one of the vigorous healthcare related pathogens and for it to acquire Carbapenem resistance, is an additional problem. Carbapenem resistance Pseudomonas aeruginosa (CRPA) became a global problem as it was discovered nearly everywhere in the world and proved to be responsible for multiple healthcare outbreaks. The pathogen has shown molecular malleability by its ability to gain different mechanisms for CR. This issue has worsened the clinical impact of healthcare in the affected places and is expected to become more worse and widely distributed unless solutions will be found and applied for it. This essay is giving some details about the mechanisms of CR, the epidemiology, and the clinical impact of CRPA.

II-Introduction

Carbapenems, amongst the beta-lactams, are the most effective antibiotics against both Gram-negative and Gram-positive bacteria presenting a broad spectrum of antibacterial activity. Their exceptional molecular structure is characterized by the existence of a carbapenem together with the beta-lactam ring which grant extraordinary stability against the majority of beta-lactamases (enzymes that inhibit beta-lactams). For these reasons (being a wide spectrum and resistant to degradation by most bacterial beta-lactamases) and combined to it, that it presents fewer side effects than most last line anti-biotics (e.g. polymyxins), Carbapenems are considered the most dependable, last line treatment for bacterial infections. Consequently, the appearance and fast worldwide spread of resistance to carbapenems, especially among Gram-negative bacteria (Pseudomonas aeruginosa is one of the most important), is considered a global problem that needs worldwide concern and plan for management (1).

Pseudomonas aeruginosa is a Gram-negative-rods, that is commonly associated with healthcare infections mainly among burn patients, ICU patients, and other vulnerable immunosuppressed patient groups. Those infections can lead to significant morbidity and mortality. The pathogen has intrinsic resistance to several classes of antibiotics, thus therapeutic options for its infections is limited (2). The problem is getting more complicated by excluding carbapenems from the treatment list of P. aeruginosa infections due to resistance problems. P. aeruginosa has shown different mechanisms for carbapenems resistance. The essay is a trail to illuminate part of the problem of CR P. aeruginosa

III-Carbapenems Resistance mechanisms

Pseudomonas aeruginosa is intrinsically resistant to several classes of antibiotics (2), but carbapenem is not one of them. Resistance to carbapenem in P.aeruginosa is an acquired type. Though the pathogen can acquire several antibiotic resistance mechanisms, mainly three are known to be involved in and deeply studied regarding carbapenems resistance; loss of the OprD porin, overproduction of multidrug efflux pumps, and production of carbapenemases. The presentation of AmpC β-lactamases (also known as class-C β-lactamases) (1) might contribute to a low potential of carbapenem hydrolysis (though it’s not considered as carbapenemase) and its overproduction might present carbapenem resistance (1).

III-I-loss of the OprD porin

Like all Gram-negative bacteria, the outer membrane of Pseudomonas aeruginosa is consisting of a semipermeable membrane that apply a control on the entrance of all molecules including antibiotics. However, this permeability differs from one kind of bacteria to another (The permeability of P. aeruginosa outer membrane is only 8% of its analogue in E. coli). The entrance of nutrients and other materials is controlled by a group of water-filled protein channels called porins. These channels are essential for the physiology of the pathogen due to their role in importing the substances needed for metabolism. However, they are also holding a problem by them having an affinity for some hydrophilic antibiotics, such as beta-lactams, tetra-cyclins, some fluoroquinolones, and aminoglycosides, allowing them to pass and harm the bacterial cell. Complete deletion or reduction of one or more of these porins was found to reduce the susceptibility of P. aeruginosa to some antibiotics including carbapenems (3).

The P. aeruginosa OprD is a substrate-specific porin that enables the diffusion of small peptides, basic amino acids, and carbapenems (especially imipenem) into the cell. Some studies have found a connection between the reduction or elimination of OprD and carbapenem resistance. A study in France by Fournier et al proved the absence of Porin OprD in 94 (86.2%) strains from a total of 109 imipenem-non-susceptible strains of P. aeruginosa(4). In a Turkish study by Agah Terzi et al oprD mRNA levels were decreased in 7 of 9 CRPA isolates evaluated (3).

OprD-mediated resistance occurs as a result of decreased transcriptional expression of oprD and/or mutations that result in loss of function and disrupted protein activity. Several mechanisms resulting in decreased transcriptional expression of oprD include (i) disruption of the oprD promotor (ii) co-regulation with trace metal resistance mechanisms (iii) premature termination of oprD transcription, (iv) decreased transcriptional expression and (v) salicylate-mediated reduction (3).

III-II-Multidrug efflux pumps

Efflux pumps are one of the most important mechanisms involved in antibiotic resistance in P. aeruginosa. They confer active expulsion of antibiotics (including carbapenems) out of the periplasmic space after their entrance. Efflux pumps are tripartite systems that are composed of a cytoplasmic membrane protein (resistance-nodulation-division family) function as a transporter, an outer membrane porin component act as a channel, and a membrane fusion protein presumed to link the two membrane proteins (1). Elevated expression of genes encoding multi-drug efflux pumps frequently produces high levels of antibiotic resistance in Gram-negative bacteria especially when it’s interplayed by the overproduction of AmpC β-lactamases (5).

Among these efflux systems in P. aeruginosa, are MexAB-OprM and MexXY-OprM which are involved in both intrinsic and acquired resistance, while MexCD-OprJ and MexEF-OprN are involved only   in acquired resistance (5). In a study done at 2002 by the Department of Microbiology, Kyoto Pharmaceutical University on the first three, MexAB-OprM, MexXY-OprM, and MexCD-OprJ, they found that strains with overexpression (of each one separately) has reduced susceptibility to all penems (as well as to norfloxacin and tetracycline) which suggest that all of the efflux systems tested extrude penems (a close cousin to carbapenems) They also concluded (from comparing the MIC for different strains that MexAB-OprM pumps expels penems more effectively than it does with tetracycline and norfloxacin, and that the extrusion power of MexAB-OprM for penems is higher than that of MexCD-OprJ and MexXY-OprM (5). Similar results for MexAB-OprM rule in CR was found in a study by the Department of Laboratory Medicine of Anhui Medical University in China in 2016 (6).

III-III-Production of a carbapenemases

This is an enzyme-mediated kind of resistance to carbapenems that involve the production of types of beta-lactamases that are able to inactivate carbapenems together with other beta-lactam antibiotics and therefore called carbapenemases (1). This type of resistance is the most important (clinically) because carbapenemases degrade all or nearly all beta-lactam antibiotics, therefore, limit the choices for treatment of P. aeruginosa (and other resistant microorganisms) infections. Carbapenemases are encoded by genes that are transferred horizontally by transposons or plasmids and are commonly associated with genes encoding other resistance mechanisms (1).

The most common carbapenemases produced by P. aeruginosa are Metallo-beta-lactamases (MBLs), also known as class-B carbapenemases (Ambler classification of β-lactamases).  MBLs are able to degrade all beta-lactams excluding aztreonam. They are not affected by beta-lactamase inhibitors like tazobactam, boronic acid, and clavulanic acid. They bear zinc in their active center, therefore, they can be inhibited in vitro by metal chelators, such as ethylenediaminetetraacetic acid (1). The genes responsible for producing MBLs are parts of an integron structure and are encoded on large transferable plasmids. Therefore, P. aeruginosa producing MBLs often present resistance to other groups of antimicrobials, which can be transferred to various types of bacteria (7)

Several types of MBLs have been recorded in P. aeruginosa strains. These include  IMP, VIM (VIM-2 most common), NDM, and SMP. The first three types are among the five most effective carbapenemases (named the big 5), in terms of carbapenem hydrolysis and geographical spread (1).

In a study done at 2006 in Calgary, Canada, they found that from 185 MBL positive P. aeruginosa 178/185 (96%) were positive for blaVIM genes and only 7/185 (4%) were positive for blaIMP genes (The encoding genes for IMP and VIM MBLs)(7). In another study in Venezuela, genes encoding VIM-2 MBL was found in all 17 CRPA strains, isolated in four hospitals in southern and eastern Venezuela, between 2007 and 2010 (8). In a study done in Iran, from 169 recorded carbapenem-resistant P. aeruginosa isolates, 26 were found to carry blaIMP and only one with blaVIM (9).

In a recent study, done in Iraq and published only in Nov. 2018 in the Asian Journal of Pharmaceutics, they found among 27 MPLs P. aeruginosa isolates, 25 were confirmed as blaVIM producer. Though OXA-48 is not common among CRPA, in the same previous study, it was found that among the same 27 isolates, 26(96%) were confirmed as blaOXA-48producers (16). This proves also that CRPA can join more than one carbapenemase producing gene in the same isolate (10).

IV-Epidemiology

Pseudomonas aeruginosa producing MetalloB-lactamases was first described from Japan in 1991 and since that time have been reported from various parts of the world including Europe, Asia, Australia, South America and North America (7). The pathogen was also proved to be involved in multiple outbreaks in tertiary centers from different parts of the world (7).

IV-I- Geographical distribution and Molecular Epidemiology

The following three maps and one table from the European Antimicrobial Resistance Surveillance Network (EARS-NET) are showing the epidemiological distribution and numbers of invasive CRPA in Europe in the years 2013, 2015 and 2017. From the maps, it can be seen that the situation in Ireland is still much better than the majority of the European countries. Though the table is confirming this point, we can notice that the number of invasive isolates in Ireland presents a successive increase in the period 2014-2017(11).

Map-1 Pseudomonas aeruginosa. Percentage of invasive isolates with carbapenems resistance, by country, EU/EEA countries, 2013 (11).

Map-2 Pseudomonas aeruginosa. Percentage of invasive isolates with carbapenems resistance, by country, EU/EEA countries, 2015 (11).

Map-3 Pseudomonas aeruginosa. Percentage of invasive isolates with carbapenems resistance, by country, EU/EEA countries, 2017(11).

Table-1 Total number of invasive Pseudomonas aeruginosa isolates tested (N) and percentage with resistance to carbapenems (%R), including 95% confidence intervals (95% CI), EU/EEA countries, 2014 to 2017(11).

In the United States, in a Surveillance for CRPA, that involved five Sites, in 2015, for a period of 4 months, 384 (9.1%) of Pseudomonas aeruginosa isolates (N=4243) were carbapenem-resistant. Resistant percentages ranged from 4.6% in Oregon to 12% in Georgia. 115 (42.0%) were female, median age was 66 years. Death occurred in 27 of 289 (9.3%) case. It was concluded in this surveillance that CRPA cases might be greater than that of CR-Enterobacteriaceae and CR-Acinetobacter. (12).

In Japan, in a study published at 2005, for evaluation of P. aeruginosa isolates collected from 60 Japanese hospitals from geographically diverse regions throughout the country, eighty-eight (15%) of 594 isolates were not susceptible to imipenem, among them 41 (47%) were not susceptible to meropenem and 1.9% was Metallo-β-lactamase-producing P. aeruginosa strains. (13).

In a similar study involved 17 hospitals in 12 cities in Poland between 2002 and 2004, 100 % of 38 imipenem-non-susceptible isolates were MBLs producers and all of them was proved positive by PCR for the presence of bla_VIM genes(14).

In Greece, thirty-six from fifty-eight imipenem non-susceptible Pseudomonas aeruginosa strains was positive for VIM-type Metallo-β-lactamase genes. The isolates were collected during May 2001 in 15 Greek hospitals(15).

In Italy, 6.5 % of 383 randomly collected Pseudomonas aeruginosa isolates, collected during 1999-2002 from three geographically distinct hospitals within Italy: Genoa (Northern Italy); Rome and Catania (Sicily), were MPLs (VIM-1 or IMP-13) producers. VIM-1-producing isolates were found at all sites, whereas IMP-13-producing isolates were only found in Rome(16).

The previous studies have shown that the emergence of MBL-producing P. aeruginosa (especially VIM-2 producing strains) occurs in different parts of the respective countries simultaneously and seemingly independent. The situation in Canada was much different according to a study done by the University of Calgary in Alberta/Canada and published in 2006. The MBL-producing P. aeruginosa have only been reported in the Calgary Health Region and do not likely present in other parts of the country(7). The study also correlated (through Molecular typing) the MBL-VIM producers to an outbreak happened during April to December 2003, in Calgary in an ICU of acute care center which then spread to the bone-marrow-transplant department, causing a similar outbreak during January to May 2004.

IV-II-Risk Factors

In a systemic review for P. aeruginosa data between 1987 and 2012, among nine factors found to be risk factors for carbapenem resistance, carbapenem use showed the strongest pooled odds ratio. The nine risk factors were, in order of statistical significance, (i)carbapenem use, (ii) medical devices, (iii) other antibiotic use, (iv) ICU admission, (v) quinolone use, (vi) underlying diseases, (vii)vancomycin use, (viii) patient characteristics, and (ix) length of hospital stay(17). (here refs for outbreaks to be added at the end). In a Spanish study by Cobos-Trigueros et al (2015) previous exposure to carbapenems was independently associated with carbapenem resistance (18).

In the surveillance made in the USA, the most common underlying condition for acquiring P. aeruginosa pulmonary infection was a chronic pulmonary disease (98 of 272).  The most frequent healthcare risk factor was hospitalization in the prior year (208 of 253; 82.2%), though 23 (7.8%) of the cases weren’t associated with any identified healthcare risk factors (12).

V-Clinical Impact.

Multidrug-resistant organisms, in general, have a negative clinical impact on several patient factors including mortality rate, length of stay in the hospital, treatment costs and some others. CRPA is not an exception from that.

In a study made in China 2018, mortality of CRPA group was compared to carbapenem susceptible P. aeruginosa group the results were12.6% vs 7.8%. The same study found a difference also in the length of stay, LOS after culture, total hospital costs (per patient), daily hospital cost, and all the results were against CRPA patients (19).

Comparable results were recorded in a study for meropenem resistant cases, in Kentucky, USA, in 2016. Increased number of deaths (28.1% vs 8.9%) and length of stay (median increase of 4 days). In addition to that, over a 40 % increase in the percentage of patients who required ICU admission(20).

A striking finding in a study made in Pennsylvania, USA was that a group of CRPA bloodstream isolates at one center, despite their MDR phenotypes, retained sensitivity to antipseudomonal beta-lactams, fluoroquinolones, and aminoglycosides. Consequently, that 92 % were treated with at least one antibiotic that was active against CRPA, without a need to resort to last-line agents such as colistin. Despite the use of treatment regimens that were active against the pathogen, mortality rates in this study were still 19% and 30% at 14 and 30 days, respectively. (21).

In a systematic review and meta-analysis by Zhang et al, published in 2016, reviewing PubMed and Embase databases up to April 2015 the results that patients with CRPA were at a higher mortality risk compared with those with carbapenem-susceptible (22).

VI-Conclusion

Castles are falling down one after another. Nearly all (if not really all) known antibiotics have a recorded resistance against them by one or more micro-organisms. In case carbapenem and other last line, antibiotics are lost as a treatment, P.aeruginosa and other MDRs infections will be undefeatable killers that millions of patients have to face while unshielded. Analyzing the problem, knowing its origin, mechanisms, distribution is an opening to find the way for a proper solution.

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