Microbiological testing must master the three major mechanisms of drug resistance

Three major resistance mechanisms that must be mastered in microbiology testing

Do you know what microbiology testing is? Do you know about microbiology testing? Here is my knowledge of the three major resistance mechanisms that must be known for microbiology testing, welcome to read.

A variety of enzymes that produce inactivated antibiotics

1, β-lactamase (β-lactamase)

β-lactam antibiotics all *** with a core β-lactam ring, and its basic mechanism of action is to bind to the bacterial penicillin-binding protein, thereby inhibiting the synthesis of the bacterial cell wall. The production of β-lactamase is the main cause of bacterial resistance to β-lactam antibacterial drugs. Bacteria produce β-lactamases that bind to and open the β-lactam ring with the help of serine active sites in their molecules, leading to drug inactivation. More than 300 β-lactamases have been reported so far, and in 1995 Bush et al. classified them into four types: type 1 is cephalosporinase that is not inhibited by clavulanic acid; type 2 is β-lactamase that can be inhibited by clavulanic acid; and type 3 is metallo β-lactamase that is not inhibited by all β-lactamase inhibitors ( requires Zn2+ activation). It can be inhibited by ethylenediaminetetraacetic acid and P-chloromercuribenzate; type 4 is a penicillinase not inhibited by clavulanic acid. Common clinical β-lactamases are ultra-broad-spectrum β-lactamases, cephalosporinases (AmpCases), and metalloenzymes.

(1) Extended-Spectrum β-lactamases (ESBLs)

ESBLs are a class of β-lactamases capable of hydrolyzing penicillins, cephalosporins, and monocyclic antibiotics. type 2 β-lactamases in the Bush typology, and their activity can be inhibited by certain β-lactamase inhibitors (rod acid, sulbactam, tazobactam).ESBLs are mainly composed of common β-lactamase genes (TEM-1, TEM-2 and SHV-1, etc.) mutated, and their drug resistance is mostly mediated by plasmids. Since the first discovery of ESBLs in Germany in 1983, more than 90 TEM-type ESBLs and more than 25 SHV-type ESBLs have been reported, and TEM-type and SHV-type ESBLs are mainly found in Klebsiella pneumoniae and Escherichia coli, as well as in Aspergillus, Providencia and other Enterobacteriaceae.

In recent years, with the widespread use of third-generation cephalosporins in China, the detection rate of ESBLs-producing bacteria has been increasing year by year, and the NCCLs stipulate that all clinical isolates of Escherichia coli and Klebsiella pneumoniae should be monitored to see if they are ESBLs-producing strains; and if they are produced, regardless of the results of the in vitro sensitization to the third-generation cephalosporins and aminotriazolam, resistance to the third-generation cephalosporins and aminotriazolam should be reported. In addition, ESBLs strains not only have a high resistance rate to β-lactam antibiotics, but also to aminoglycosides and quinolones with a resistance rate of about 60%, therefore, when clinically encountering infections caused by ESBLs, it is recommended that compound antibiotic preparations containing β-lactamase inhibitors or imipenem be preferred; there is a controversy about fourth-generation cephalosporins such as cefepime , according to the PK/PD theory of antimicrobials, the dose and dosing interval should be changed appropriately. So that the blood concentration exceeds the bacterial MIC for 40% of the dosing interval or more, may be effective.

(2) Cephalosporinase (AmpCase) session Bush classification of type 1 (type I) β-lactamases.

It is usually divided into AmpC β-lactamase produced by chromosome-mediated production and AmpC β-lactamase produced by plasmid-mediated production, the former produces bacteria such as Enterobacteriaceae inguinalis and Pseudomonas aeruginosa, the latter is mainly produced by Klebsiella pneumoniae and Escherichia coli. AmpCase acts on most penicillins, first, second and third generation cephalosporins and monocyclic antibiotics. Fourth-generation cephalosporins and carbapenems are not affected by the enzyme. The enzyme cannot be inhibited by β-lactamase inhibitors.There are 2 possibilities for AmpC β-lactamase production: ① Temporary high level production in the presence of an inducer, and a subsequent decrease in enzyme production when the inducer is not present.The third generation cephalosporins, rotenoids, and carbapenem antibiotics are strong inducers of the inducible AmpC enzyme;② Mutations in the genes controlling enzyme expression on the chromosome resulted in the continuous stable high level expression of AmpCase. Infections caused by highly AmpCase-resistant bacteria have a high mortality rate.

Virtually all Gram-negative bacteria produce chromosome-mediated AmpC cephalosporinase, in most cases at low levels; in Enterobacteriaceae, Citrobacter, Serratiaceae, and Pseudomonas aeruginosa, production is induced at a high frequency, and is often in highly productive mutant strains. When the clinical appearance of the above bacterial infections, the first few days of three-generation cephalosporin treatment is sensitive, and the subsequent occurrence of resistance, we can suspect that the high production of AmpC enzyme bacterial infection, four-generation cephalosporins and carbapenem antibiotics are not subject to the specific influence of the clinical options. Enzyme inhibitor-containing combinations should not be used to treat infections with AmpCase-producing strains.

(3) Metalloβ-1actamase

Most β-lactamases have serine residues in the active site, but there are a small number of enzymes with metal ions in the active site. The first enzyme found to have a metal ion as its active center was cephalosporinase produced by Bacillus cereus, which was inhibited by EDTA, and a variety of bacteria producing this type of enzyme have been found around the world since then. 1988 Bush first named the enzyme metalloβ-1actamase, or metalloenzyme for short. Metalloβ-1actamase is resistant to β-lactamase inhibitors and can hydrolyze almost all β-lactam antibiotics (including imipenem). The enzyme has been found in Aeromonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia, where imipenem resistance in Stenotrophomonas maltophilia is chromosomally mediated, and plasmid-mediated mutant strains in Pseudomonas fragilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa have been reported in Japan. The metallo-β-lactamase type IMP-1 produced by Serratia marcescens, which can move on spliceon-like intl3, has spread to Pseudomonas aeruginosa, Klebsiella pneumoniae, and alkali-producing bacilli. The metalloenzyme hydrolyzes carbapenems and recently developed fourth-generation cephalosporins. Metallo-beta-lactamases have the potential for wide dissemination and have hydrolytic activity against almost all beta-lactam antibiotics, making them the most potent beta-lactamase- known to date.

2. Aminoglycoside-modifying enzymes (or passivating/inactivating enzymes)

In the mechanism of bacterial resistance to aminoglycoside antibiotics, modifying enzyme-mediated resistance is the most prevalent, and enzymatically-modified aminoglycoside antibiotics are unable to interact with ribosomal targets, and therefore lose their antibacterial activity. Modifying enzymes mainly include acetyltransferases, phosphotransferases and nucleoside transferase. The mechanism of action of the three types of aminoglycoside-modifying enzymes is different: acetyltransferase (AAC) modification is dependent on the N-acetylation of acetyl-coenzyme A: phosphotransferase (APH) modification is dependent on the O-phosphorylation of ATP; and nucleotidyltransferase (ANT) modification is dependent on adenylation of ATP. In Gram-negative pathogenic bacteria, the most common aminoglycoside-modifying enzyme is AAC (6'), which acetylates aminoglycoside antibiotics at the 1-, 3-, 2'-, or 6'-position. Today 16 genes encoding AAC(6') have been identified. Pseudomonas aeruginosa and Enterobacteriaceae tend to produce AAC(3), AAC(6'), ANT(2''), and APH(3'); staphylococci and Enterococcus faecalis frequently produce ANT(4' )(4'') or the bifunctional AAC(6')/APH(2"). Staphylococcal `resistance to gentamicin, kanamycin, and tobramycin and high gentamicin resistance in enterococci are usually mediated by bifunctional enzymes that are usually (but not always) encoded by transposons located on multidrug-resistant plasmids (Tn924), such as APH (3') encoded by transposon Tn5405, possessed by staphylococci (providing kanamycin, neomycin and amikacin resistance), while others are localized to chromosomes. An increasing number of strains produce 2 or more enzymes against aminoglycoside antibiotics. A common combination in the last few years has been the gentamicin-modifying enzymes ANT(2'') and AAC(3)] in combination with AAC(6'), leading to broad-spectrum resistance to gentamicin, tobramycin, netilmicin, kanamycin, and amikacin.

Aminoglycoside antibiotics have excellent antimicrobial activity against non-fermenting bacteria, Enterobacteriaceae, and some Gram-positive cocci, and have synergistic antimicrobial effects in combination with β-lactam antibiotics, which have an important role in the treatment of infections. However, due to the existence of the above resistance mechanisms, the problem of bacterial resistance is also becoming more and more serious, and it should be emphasized. It is encouraging to see that amikacin and other antibiotics against MRSA and ESBLs-producing strains still maintain a sensitivity rate of 17%-40%.

Second, change the target of drug action

1, penicillin-binding protein (PBP) changes caused by β-lactam antibiotic resistance

Penicillin-binding protein (PBP) is involved in the peptidoglycan synthesis of final stage. High molecular weight PBPs are often multimodular, with an N-terminal glycosyltransferase region and a C-terminal transpeptidase region. The active site serine in the transpeptidase region resembles the natural structure of the enzyme and can be irreversibly acylated with β-lactam antibiotics. Alterations in penicillin-binding protein (PBP) often lead to the following two clinically important resistance phenotypes.

(1) Methicillin-resistant Staphylococcus aureus (MRSA)

MRSA was first reported in the United Kingdom in the 1960s as a serious clinically drug-resistant pathogen. MRSA is a serious clinical drug-resistant pathogen first reported in the United Kingdom in the 1960s. Since the 1980s, outbreaks of MRSA hospital-acquired infections have occurred around the world and have been increasing year by year.MRSA resistance is divided into intrinsic resistance and acquired resistance.Intrinsic resistance is chromosome-mediated, and its resistance is due to the bacterium's production of a special penicillin-binding protein, PBP2a (or PBP2'), which is a protein of molecular weight of 78,000 and has a decreased affinity to beta-lactam antibiotics. PBP2a is encoded by the mecA gene, which is detected in more than 95% of MRSA strains and absent in sensitive strains. Acquired resistance is plasmid-mediated, and bacteria that acquire the resistance gene produce large numbers of β-lactamases (rather than PBPs), which slowly inactivate the enzyme-resistant penicillin and exhibit resistance, mostly critical resistance.

In the process of MRSA detection, where MRSA, regardless of its MIC value for other β-lactam antibiotics or the size of the circle of inhibition, the laboratory should be reported to the clinic as resistant to all penicillins, cephalosporins, carbapenems, carbocephalosporins, and β-lactams - enzyme inhibitor composite preparations, so as not to mislead the clinical use of drugs. The treatment of MRSA infection is one of the very difficult clinical problems, the key is that it is multi-drug resistant to many antibiotics, vancomycin is the current clinical treatment of MRSA efficacy is certain antibiotics, the application of more than 30 years have not been found resistant strains.

(2) Penicillin resistant Streptococcus pneumoniae (PRSP)

For a long time, Streptococcus pneumoniae has been highly susceptible to penicillin, with a MIC of 0.005-0.01mg/L. The first report of penicillin resistant Streptococcus pneumoniae was made in Australia in 1967. Penicillin-resistant Streptococcus pneumoniae was first reported in Australia in 1967, with a MIC of 0.5mg/L. Since then, it has been reported in many countries and regions of the world, and the rate of resistance has been increasing rapidly.The resistance mechanism of PRSP is the alteration of the penicillin-binding protein (PBP) of Streptococcus pneumoniae, which reduces its affinity for penicillin. Streptococcus pneumoniae has six PBPs: 1a, 1b, 2x, 2a, 2b, and 3, of which PBP2b is the most important, and if penicillin binds to and inhibits PBP2b that leads to bacterial lysis and death; conversely, mutations in PBP2b, where penicillin fails to produce an effect, lead to PRSP.In PRSP highly resistant strains (MIC ≥ 2 μg/m1) there can be up to four PBPs (mainly 1a, 1b, 2x, and 2b) altered simultaneously [7].

Streptococcus pneumoniae is an important causative agent of community-acquired pneumonia. Currently, the incidence of PRSP in China is around 4%, which is significantly lower than that in European countries and is also in the middle level in Asia, and the MIC is mostly less than 1 mg/L. Therefore, among the pathogenic bacteria of community-acquired lung infections, PRSP does not yet pose a serious threat, and penicillin can still be used as the first choice of therapeutic drug. However, drug resistance has no national boundaries, and the incidence of PRSP in China a few days ago was still low. The incidence of PRSP in China is still low. However, it does not mean that we should not pay attention to it, but we should further strengthen the drug resistance monitoring of PRSP. For clinical treatment of PRSP infection, cefotaxime/ceftriaxone and new quinolones (e.g., sparfloxacin) are recommended. In severe PRSP infections, vancomycin or rifampicin should be used.

2, DNA topoisomerase changes caused by quinolone antibiotic resistance

The mechanism of action of quinolones is mainly through the inhibition of DNA topoisomerase and inhibition of DNA synthesis, so as to play the role of bacteriostatic and bactericidal. Bacterial DNA topoisomerases are I, II, III, and IV, and the main targets of quinolones are topoisomerase II and topoisomerase IV. Topoisomerase II, also known as DNA promoter, is involved in the formation of the DNA superhelix, while topoisomerase IV is involved in the distribution of bacterial zygotic chromatin into zygotic bacteria. DNA procyclase is the first target site of quinolones in Gram-negative bacteria, while topoisomerase IV is the first target site in Gram-positive bacteria.

Quinolone resistance can be caused by mutations in the genes encoding either of the A and B subunits that make up DNA gyrase and the parC and parE subunits that make up topoisomerase IV. Among all the mutant types, mutations in gyrA predominate, accounting for about 80% of the cases, followed by gyrB, parC and parE mutations. Among all these mutation types, if two mutation sites were present on type II topoisomerases (e.g., on gyrA and parC), they induced much greater resistance to fluoroquinolones than if there was only one mutation site (e.g., on gyrA or gyrB), with the former being 3-4 times more common than the latter. At the same time, the phenomenon that mutations were only found in the parC gene was not observed. This may be due to the fact that the DNA gyrase is an important target site for fluoroquinolones, and alterations in the gyrA subunit may cause structural changes in the enzyme resulting in spatial barriers, preventing the entry of quinolones into the quinolone zone of action, or causing physicochemical changes that interfere with quinolone-enzyme interactions. These results suggest that the presence of mutations on gyrA is the primary mechanism for causing bacterial resistance to quinolones, whereas the parC mutation only further induces a high level of quinolone resistance in P. aeruginosa.

Alterations in DNA topoisomerases are the primary mechanism by which bacteria become resistant to quinolones, and other mechanisms of quinolone resistance include altered bacterial membrane permeability and active efflux mechanisms, which will be discussed later.

Three, the cell membrane permeability barrier and antibiotic active efflux pump

Bacteria can form an effective barrier through the obstacle of the cell wall or the alteration of the cell membrane permeability, so that antibiotics are unable to enter the cell and reach the target to play antibacterial efficacy, which is also a kind of defense mechanism formed by the bacteria in the process of evolution and reproduction. This is also a defense mechanism formed during the evolution and reproduction of bacteria. This type of resistance mechanism is non-specific and is mainly found in Gram-negative bacteria. The outer membrane of Gram-negative bacteria consists of a lipid bilayer outside the mucopeptide layer of the cell wall, and the outer layer is a lipopolysaccharide composed of tightly arranged carbon and nitrogen molecules, which prevents hydrophobic antimicrobials from entering the bacterium. In addition there are a variety of pore proteins on the bacterial outer membrane, the larger molecule is OmpF, the smaller molecule is OmpC, they can form specific channels (OprD) and non-specific channels (OprF), as nutrients and hydrophilic antimicrobial drug channels. The larger the antimicrobial drug molecule, the more negatively charged it is and the more hydrophobic it is, the less likely it is to pass through the bacterial outer membrane. Bacterial mutation loss of a specific pore protein can lead to bacterial drug resistance, in addition, due to the lack of outer membrane protein OprF, so that the drug is not easy to pass through and produce drug resistance. For example, Pseudomonas aeruginosa specific pore protein OprD2 loss that leads to carbapenem antibiotic resistance.

Another mechanism that leads to non-specific resistance in bacteria is the presence of a bacterial active efflux pump, which pumps drugs that enter the bacterial body out of the membrane, thus evading the action of antibiotics. The active efflux system results in the acquisition of resistance by the cell due to its ability to actively pump out of the cell a wide range of antimicrobial drugs that have entered the cell in a specific manner. For example, the multidrug efflux pump AcorAB-TolC system in Escherichia coli can lead to bacterial resistance to a wide range of structurally unrelated drugs including tetracyclines, chloramphenicol, erythromycin, β-lactams, rifamphenicol, fluoroquinolones, oxidizing agents, organic solvents, and basic dyes. The active exocytosis of the MexAB-OprM system of P. aeruginosa is also one of the important factors contributing to the inherent multi-drug resistance of P. aeruginosa.

The mechanism of membrane resistance in bacteria is mainly manifested in the multidrug resistance of P. aeruginosa. Pseudomonas aeruginosa encompasses almost all bacterial drug resistance mechanisms, including membrane resistance, and its drug resistance has become one of the more difficult problems in the treatment of current infections, which is particularly worthy of attention and research.