Gonorrhea is the main type of STD prevalent in China. Although the increase in the number of patients with other STDs has reduced the proportion of gonorrhea in the overall STD patients from 65.2% in 1991 to 33.3% in 2000, the total number of gonorrhea patients has increased from 114,000 to 286,000, and the incidence rate has increased from 10.09/100,000 to 22.92/100,000.
I. Current status of gonococcal drug resistance
Antibiotics are still the main drugs for treating gonorrhea, but because the medical market for treating venereal diseases in China is not standardized, the number of resistant strains of gonococci has increased, such as the resistant strains of gonococci to penicillin has reached 68.3%, and the resistant strains of tetracycline has reached 92.6%, so these two drugs have been withdrawn from the treatment program of gonorrhea. Ciprofloxacin, a quinolone drug, has only been used for gonorrhea treatment in recent years, but the proportion of resistant strains has risen rapidly. The National STD Leprosy Control Center Gonococcal Drug Resistance Surveillance Collaborative Group reported that during the 6 years from 1995 to 2000, the resistant strains of gonococci to ciprofloxacin were 15.5%, 13.5%, 28.5%, 52.3%, 78.2% and 85.2%, respectively. Among the 4188 gonococcal strains tested, 2232 strains (53.3%) were resistant to ciprofloxacin. Many cases of gonorrhea treatment failure with ciprofloxacin were also found in the clinic.
II. The problem of gonococcal drug resistance
More and more bacteria encountered in the clinic are resistant, so more and more antibiotics do not work. The reason for this is that the genes of bacteria have changed, and there are many ways to change, the simpler the organism, the easier it is to mutate.
1.Commonly used antibiotics
According to the chemical structure, the commonly used antibiotics can be divided into: β-lactams, aminoglycosides, macrolides, polyenes, tetracyclines, sulfonamides, quinolones and so on.
Antibacterial drugs inhibit or kill microorganisms mainly through five action sites: 1. Inhibit the synthesis of bacterial cell wall, the drugs with selective toxicity to bacteria are β-lactams (penicillins, cephalosporins, carbapenems), glycopeptides (vancomycin, teicoplanin); 2. Inhibit bacterial protein synthesis, the drugs with selective toxicity are macrolides, tetracyclines, chloramphenicol Inhibit folic acid synthesis, selective drugs such as sulfonamides, methotrexate; 4. Affect nucleic acid metabolism, drugs such as quinolones, rifampin, metronidazole, selective toxicity is poor; 5. Destroy cell membrane structure, drugs such as amphotericin B, polymyxin, selective toxicity is poor.
2.Classification of gonococcal drug resistance
Genetic resistance (natural resistance): chromosomal resistance (chromosomal-mediated), extrachromosomal resistance (plasmid-mediated)
(plasmid-mediated) and non-genetic resistance (acquired resistance): mostly plasmid-mediated.
3. Mode of transmission of gonococcal drug resistance
There are several ways of plasmid-mediated and chromosome-mediated transmission of genes from one bacterium to another in gonococci.
(1) transformation (transduction).
This refers to the entry of a generally exogenous gene into the bacterium and its integration into the chromosome of the host bacterium. All gonococci can absorb, integrate, and express DNA from other organisms, so all gonococci have the ability to transform. The ability to transform is significantly higher in gonococci with germinal hairs than in germ-free gonococcal variants. Transformation can occur at any time during the growth of a hairy gonococcus. Gonococci can be transformed by plasmid and chromosomal DNA;
(2) Binding.
Binding is another form of exchange of gonococcal genetic information. DNA is transferred and exchanged when gonococci come in contact with each other. The process of pair binding and exchange of genetic information is mediated by a very large, 24.5 megadalton plasmid. This plasmid is capable of transferring its resistance plasmid between different gonococcal strains. Transfer of resistant plasmids can occur not only between gonococci, but also between gonococci and other types of bacteria, including other Neisseria species as well as E. coli. Therefore, binding is very important for the spread of antibiotic-resistant plasmids in vivo, and it is very meaningful for studying the mechanism of plasmid exchange;
(3) transduction (conjugation), translocation or transposition.
Transduction refers to the transmission of DNA from the donor bacterium to the recipient bacterium via phage, which is then embedded in the DNA of the recipient bacterium by recombination. Translocation or transposition refers to the exchange of a segment of DNA sequence between plasmids or between plasmids and chromosomes, resulting in drug resistance. In the study of possible mechanisms of exchange of gonococcal genetic material, only two modalities, translocation and conjugation, have been identified so far.
Drug resistance in gonococci can be caused by chromosomes or by drug-resistant plasmids. With the development of genetics and molecular biology, the mechanism of chromosomal-mediated drug resistance in gonococci is becoming more and more clearly understood. It is now believed that there are two types of chromosomal-mediated drug resistance: single-step mutations in the genes of drug target loci, and mutations involving several genes of drug target loci, with the combined mutations of different loci determining the degree and mode of drug resistance.
Current research on chromosomal-mediated resistance has focused on mutations at multiple loci, which include penicillin-binding protein genes (penA, penB), DNA helicase A gene (gyrA), C-subunit gene of topoisomerase IV (parC), multiple transferable resistance system genes (MtrR, MtrC, MtrD, MtrE) and pore protein genes ( porin, pIA/pIB), etc. The results of these studies were obtained by using the “functional cloning method” to first clone genes related to antibiotic target sites or metabolic resistance, such as penA, penB, gyrA, parC, Mtr (MtrR, MtrC, MtrD, MtrE) and porin ( pIA/pIB), and then further analyze the altered sequences of the corresponding genes in strains with emergent resistant phenotypes.
Many studies have demonstrated the existence of multiple resistance mechanisms in fluoroquinolone-resistant gonococcal strains. Therefore, gonococcal drug resistance may be a multigene controlled trait. However, the number of genes that play a role in gonococcal resistance and the existence of more important resistance-associated genes are topics that deserve to be studied in depth.
Gonococci have different resistance rates and resistance mechanisms to commonly used antibiotics: the main mechanism of resistance to penicillin is the production of β-lactamase; the main mechanism of resistance to tetracycline is the reduced permeability of the cell membrane to the drug; the main mechanism of resistance to macrolides and macrolides is the alteration of the target site of action; the main mechanism of resistance to fluoroquinolones is the mutation of gyrA and parC genes; the resistance to third generation The mechanism of reduced susceptibility or resistance to third-generation cephalosporins has not been clarified, but it is closely related to genes such as penB and penA.
Three, gonococcal drug resistance mechanism
1.Resistance plasmids
The specific drug resistance of gonococci can be caused by the genetic alteration of chromosome and plasmid, and gonococci are generally classified into the following types according to their drug resistance: PPNG plasmid-mediated penicillinase-producing gonococci; TRNG plasmid-mediated tetracycline-resistant gonococci; PPNG/TRNG plasmid-mediated gonococci resistant to both penicillin and tetracycline; CMRNG chromosome-mediated gonococci resistant to both penicillin and tetracycline; and CMRNG chromosome-mediated gonococci resistant to both penicillin and tetracycline. CMRNG chromosome-mediated gonococci resistant to both penicillin and tetracycline; QRNG chromosome-mediated gonococci resistant to quinolones.
Penicillin-resistant gonococcal strains with β-lactamases were first isolated in 1976, and studies have shown that this resistance is mediated by plasmid-mediated resistance to penicillin and ampicillin. PPNG is currently disseminated worldwide, with 50% or more of PPNG in many parts of Africa and Asia, posing considerable difficulties in the treatment of gonorrhea; β-lactamase negative by chromosomally mediated resistant Neisseria gonorrheae strains (CMRNG) were isolated in the United States in 1983; plasmids were first identified in 1985 TRNG is resistant to tetracycline, dimethylaminotetracycline and doxycycline. Local outbreaks of PPNG, CMRNG and TRNG have been confirmed to be a nutritional serotype, and control should focus on controlling resistant strains.
Human research on gonococcal plasmids began in the 1970s. 2.6 MD of cryptic plasmids were first detected from a case of gonorrhea by Englkirk in the United States in 1973, followed by penicillin-resistant plasmids (3.2-3.4 MD for the African type, 4.4-4.7 MD for the Asian type and 3.05 MD for the Toronto type), highly tetracycline-resistant plasmids (25.2 MD ) and splice type plasmids (24.5 MD). It was shown that the splice type plasmids were closely related to the dissemination of drug-resistant plasmids between strains, and were able to transfer their drug-resistant plasmids between different gonococci or even between gonococci and other bacteria.
The 25.2MD plasmid carries the TetM gene, which mediates high levels of tetracycline resistance. It was thought that this plasmid was formed by the acquisition of the TetM gene from the 24. 5MD plasmid, but Gascoyne et al. reported that the restriction enzyme profiles of the two plasmids were significantly different, and it is now believed that the 24. 5MD plasmid and the 25. 2MD plasmid are not homologous.
The 24.5MD plasmid is closely related to the 4. 4MD penicillin-resistant plasmid, and Lind et al. reported that 81% of PPNG with the 4. 4MD plasmid also had the 24.5MD plasmid, suggesting that the 24.5MD plasmid may be related to the delivery of the 4. 4MD plasmid. Drug-resistant plasmids can be transferred between strains by both transformation and splicing. In transformation experiments, Graves found that only homologous fragments could be taken up, and therefore plasmid transformation requires the identification of specific DNA sequences. Specific receptors may exist on the surface of gonococci. Further studies of plasmid transformation will help to better understand the mechanisms underlying the widespread spread of drug-resistant plasmids.
2.6MD cryptic plasmids can be detected in most strains and their significance is unclear, Korch et al. demonstrated that cryptic plasmids of gonococci can be integrated into bacterial chromosomes (including plasmid-free strains) and that recombinant bacteria can undergo antigenic changes such as hair and outer membrane proteins.
There is a significant regional variation in plasmid carriage in gonococci. In addition to regional differences in gonococcal plasmid carriage, there is a high diversity of penicillin-resistant plasmids. The relationship between these plasmids and gonococcal drug resistance is unclear.
2. Research on serological typing of gonococci
Different methods have been used to actively understand the distribution, frequency, origin and epidemiological characteristics of drug-resistant strains.
tapsall et al. studied the phenotypic characteristics of fluoroquinolone susceptibility reduced (76 strains) and drug-resistant (21 strains) gonococci isolated from 97 gonorrhea patients in Sydney from 1991 to 1995. The results of trophic/serotypic (A/S) typing showed that 97 gonococcal strains belonged to 27 A/S types, of which 10 strains (10%) were serotype IA and belonged to 5 different A/S types. 87 strains were serotype IB and belonged to 22 different A/S types. All drug-resistant strains belonged to IB serotypes, including 14 ciprofloxacin MIC 8 μg to 16 μg/ml gonococci with 6 different IBA/S types. The diversity of phenotypes suggests that different subtypes of gonococci are capable of developing fluoroquinolone resistance.
Kam et al. conducted a comparative study of the serotypes and antibiotic susceptibility profiles of 69 resistant strains isolated from patients failing treatment with ofloxacin in Hong Kong from January 1991 to January 1995 and 143 other sensitive strains. 69 resistant strains belonged to 21 serotypes, the majority (67/69) were serotype IB, with BoP and Bpy predominating, pointing to 92.7%. Most IA and other IB serotypes decreased during quinolone resistance selection.
Thirty-one strains of fluoroquinolone-resistant gonococci isolated in Japan belonged to 11 serotypes, all of which were serogroup WII/WIII (i.e., serotype IB). Various subtypes of gonococci showed alterations in gyrA and parC associated with quinolone resistance. These studies suggest that the emergence of fluoroquinolone-resistant gonococci in Australia, Hong Kong, and Japan is due to polyclonal selection of strains with altered gyrA and parC genes in vivo, and that certain IB serotype strains appear to be more susceptible to selection.
A synergistic agglutination test was applied to study changes in gonococcal serotyping in the Heidelberg region of Germany between 1985 and 1990. The serological typing of 649 gonococcal specimens showed that 8 and 26 serotypes were detected among the 24 known specific protein A (PIA) and 31 specific protein B (PIB), respectively, with the 6 most common serotypes being IB-3 (19.0%), IB-4 (16.0%), IB-2 (15.7%), IB-1 (13.1%) , IA-1/2 (11.7%) and IA-6 (3.2%). In addition, we found an unknown PIB with seropositive patterns of 3C8, 1F5, 2D4. concluded that the serotypes of gonococci in this region are still predominantly IB.
3. Inactivating enzymes that produce antibiotics
Some clinically used β-lactam antibiotics such as penicillin (PC) and cephalosporin (CS) contain β-lactam. β-lactam antibiotics selectively act on the bacterial cell wall, β-lactam ring can bind to the synthesis of mucopeptide transpeptidase, so that the transpeptidase is still inactive, and then inhibit the synthesis of the cell wall, and this type of drug is less toxic to animal cells without cell wall, so β-lactam Therefore, β-lactams are commonly used in the clinical treatment of gonorrhea.
Some gonococci can develop resistance to β-lactam antibiotics by producing β-lactamases, which are different types of enzymes that use the β-lactam ring as a substrate for degradation, which can reduce or eliminate the antibacterial effect of antibiotics.
The mechanism of action: the active center of penicillinase or cephalosporinase is serine in the protein polypeptide chain, which acts as a nucleophile and the carboxyl group of the β-lactam ring, forming an acylase intermediate, which leads to the inactivation of the β-lactam ring under the action of H2O molecules, which is an important reason for the resistance of gonococci to β-lactam antibiotics.
4.Changing the target site of existing action
This mechanism includes penicillinbind-ingproteinsPBPs, DNA helicase A subunit (gyrA) and topoisomerase C subunit (parC).
4.1 Penicillinbind-ingproteinsPBPs
The resistance caused by PBPs is plasmid-mediated, and the resistant plasmid is able to encode a synthetic β-lactamase that disrupts the structure of penicillin.
PBPs are some enzymes located on the cell membrane, including: peptidoglycan transpeptidase, glucose transpeptidase, carboxypeptidase, which play an important role in maintaining the stability of the bacterial cell wall, and are also the main target sites of β-lactam antibiotics, usually pathogenic gonococci have relatively simple PBPs pattern, according to the protein electrophoresis bands can be divided into three types, respectively, PBP1 (87000KD) PBP1, PBP2 (59000KD), PBP3 (44000KD), of which PBP1 and PBP2 are important antibiotic target sites. β-lactam antibiotics kill gonococci by specifically binding to PBPs on the gonococcal cell membrane, interfering with the synthesis of cell wall peptidoglycan and affecting cell wall synthesis. In sensitive gonococcal strains, PBP2 binds 100% to penicillin; in resistant gonococcal strains, PBP2 binds only 25% to penicillin. However, the change in PBP2 affinity was the result of a mutation at the Pen A locus on the chromosome
In the mechanism of gonococcal drug resistance, it is known that ponA and PenA are genes encoding PBPs, and when ponA and PenA genes are mutated, the corresponding amino acid sequences can be changed such as the mutation of PBP1 (Leu-421→Pro) and PBP2 (Asp-345a insertion), thus affecting the binding of gonococci to β-lactam antibiotics, The rate of acylation of the cell wall by β-lactam antibiotics was reduced by 3-4 times, thus having the effect of enhancing bacterial resistance.
4.2 Alteration of DNA helicase (gyrA) and topoisomerase (parC)
Some fluoroquinolone antibiotics target the bacterial DNA helicase, a type II DNA topoisomerase that plays an important role in DNA replication, recombination and transcription. The mutation of this gene can cause alteration of the A subunit of the drug target site and affect its ability to bind to the drug, causing the gonococci to show resistance to the drug.
Yoshda et al. introduced plasmids with the gyrA gene of drug-resistant strains into normal gonococcal strains and showed that the sensitive strains were also resistant to the drugs.
In a further study by Deguchi, mutations in codons 91 and 95 were found in the gyrA gene of drug-resistant strains, resulting in the change of TCG (serine) to TTC (phenylalanine) at position 91 and GAC (aspartate) to AAC (asparagine) at position 95, as well as mutations at positions 86, 87, 88 and 91 in parC, but not in drug-susceptible strains. This study suggests that gyrA and parC locus changes are important drug resistance mechanisms in gonococci.
Belland et al. also studied the relationship between gyrA and parC in drug-resistant strains and found that mutations at the gyrA locus were the most important resistance mechanism in gonococci, and mutations at this locus could cause low and moderate levels of resistance, while mutations at parC played a secondary role in gonococcal resistance. The combination of gyrA mutation and parC mutation caused higher level of drug resistance.
4.3 Synergistic effect of DNA helicase alteration and reduction of intracellular drug-like accumulation
The reduction of intracellular drug accumulation was also involved in the development of drug resistance, but its effect was relatively small. The reduction in intracellular drug accumulation may be related to the active efflux system of the intracellular membrane. For example, a decrease in fluoroquinolone uptake and accumulation was observed in clinical isolates with reduced fluoroquinolone sensitivity. In a laboratory study of selective mutant strains, it was found that in the absence of gyrA and parC gene mutations, the mutant strain with reduced intracellular oxifloxacin accumulation had a 16-fold higher oxifloxacin MIC than the parental strain; the mutant strain with a gyrA single site mutation with reduced intracellular drug accumulation had a 128-fold higher MIC; the mutant strain with mutations in both gyrA and parC with reduced intracellular drug accumulation The MIC values of the mutant strains with mutations in both gyrA and parC with reduced intracellular drug accumulation were increased 256-fold. There was no significant difference in the characteristics of the outer membrane proteins between the drug-resistant mutant strains with and without the decrease in drug accumulation.
5. Mechanism of drug efflux
The Mtreffluxsystem plays an important role in the drug resistance mechanism of gonococci. This system determines the susceptibility of gonococci to lipid-soluble factors (HAs), including fatty acids, bile salts, and lipid-soluble antibiotics.
Doughterty found in 1986 that in addition to PPNG resistance mediated by the prc plasmid, there is another group of resistant gonococci that do not produce β-lactamases, but these strains all have variants of the Mtr gene system.
The Mtr system (mutipletransferableresistance) is a multitransmittable resistance system, which is a multitransmittable resistance manipulator consisting of a suppressor MtrR gene and three structural genes MtrC, MtrD and MtrE, which can encode the corresponding proteins (MtrR, MtrC, MtrD and MtrE), respectively. -MtrC is a membrane fusion protein; MtrD is an exocytosis protein, located on the cell membrane; MtrE is an outer membrane channel protein, located outside the cell membrane; MtrR is a regulatory protein, which inhibits the transcription of MtrCDE.
This system is very similar to the AcrAE and EnvCD protein pumps on the cell membrane of E. coli and the MexABOprK protein pump on the cell membrane of P. aeruginosa. The Mtr gene complex of gonococci is an active efflux protein pump composed of MtrC-MtrD-MtrE cell membrane proteins. The MtrC-MtrD-MtrE complex can effectively transport various structurally different antibiotics and lipid-soluble factors (HAs) out of the cell actively, and the whole process is catalyzed by lipoproteins in the cell membrane under ATPase. The gonococci actively pump out antibiotic molecules through this energy-dependent system to prevent the accumulation of drugs in the cell, and thus the antibiotics cannot reach the concentration required for their selective cytotoxic effects, so this group of gonococci can exhibit multiple resistance to antibiotics.
The regulation of multi-drug resistance in gonococci by the Mtr efflux system can be divided into two mechanisms:
1. MtrR-dependent regulation mechanism
In the MtrR-dependent regulatory mechanism, it is known that the MtrR gene has 630 bp and is located 250 bp upstream of the MtrCDE gene, which can encode a 210 amino acid protein. A mutation in the base of MtrR gene leads to a decrease in the synthesis of MtrR repressor protein and an increase in the transcription of MtrCDE gene downstream of MtrR gene, resulting in an increase of MtrCDE protein in the cell membrane, which in turn results in the active efflux of antibiotics by the Mtr efflux system of gonococci. The active efflux function of the gonococcal Mtr efflux system to antibiotics.
2. Non-MtrR-dependent regulatory mechanism
In addition, there is a 13bp palindrome (5′-AAAAA-GACTTTTT-3′) in the promoter region between the MtrR and MtrC genes, and when the last base T/A at the 3′ end of this palindrome is missing, it affects the expression of MtrR and The deletion of the last base T/A at the 3′ end of this echo structure affected the expression of MtrR and MtrC genes, thus increasing the resistance of gonococci to antibiotics in a non-MtrR-dependent regulation. It has been shown that this mechanism of gonococcal drug resistance is significantly stronger than that of MtrR-dependent regulation.
Recently, it was found that there is another system on the gonococcal cell membrane, namely Far system, which is resistant to long-chain fatty acids and some lipid-soluble factors, but some scholars found that although Far system and Mtr system act independently on various lipid-soluble factors and antibiotics, but Far system still needs to rely on the outer membrane channel protein MtrE, and will also be regulated by MtrR protein.
6.Change of cell membrane permeability
Antibiotics and other complex molecules must enter the cell through the lipopolysaccharide outer membrane channel, a channel provided by the pore protein (porin), the ability of antibiotics through this channel will be affected by its shape, size, charge, once the pore protein is mutated, the bacteria are prone to drug resistance.
There are at least three outer membrane proteins of gonococci, of which protein I is the main protein, accounting for 60% of the outer membrane proteins, and the antigenicity of protein I varies among gonococci. The antigen is stable, so it can be used to make monoclonal antibodies for serological typing of gonococci. It is expressed in two forms, PIA and PIB, and forms pores in the cell membrane. It allows water-soluble substances, other substances important for bacterial metabolism and some antibiotics to enter the cell through the cell membrane. Protein II is related to the adhesion of gonococci to human epithelial cells, leukocytes and intercellular adhesion, and has thermal modifying properties. Protein III is reductively modified, also known as Rmp, and has strong immunogenicity, cross-reacting with other Neisseria species and blocking the bactericidal effect of other antibodies.
When the expression of outer membrane proteins PIA and PIB of gonococci is decreased or not expressed at all, the amount of antibiotics entering the bacterial cytoplasm will be reduced and gonococci will show resistance to antibiotics.
7. Adaptation of gonococci and other immune evasion mechanisms
The variation of surface components include bacteriophage variation, Opa protein variation, LPS variation; using the components of the host, such as gonococci need iron ions for growth, expressing receptors that recognize transferrin and lactoferrin, using the body’s iron. Some studies have shown that the iron acquisition system of bacteria is also the basic virulence factor. There are also immune evasion mechanisms such as bacterial endosurvival and serum resistance.