Plasmid-mediated resistance to tetracyclines among Neisseria gonorrhoeae strains isolated in Poland between 2012 and 2013.

INTRODUCTION: One of two main mechanisms of resistance in tetracycline-resistant Neisseria gonorrhoeae (TRNG) is associated with the presence of TetM protein responsible for actively blocking of the tetracycline target site in the 30S ribosomal subunit. This mechanism is encoded by conjugative plasmids. The second mechanism is chromosomal in nature and due to mutations in specific genes. AIM: To determine the incidence and type of tetM determinants in TRNG strains isolated from patients presenting with gonorrhea infection to the Dermatology and Venereology Clinic in Warsaw in 2012-2013. MATERIAL AND METHODS: Tetracycline and doxycycline susceptibility was determined by E-Tests. The presence and type of the tetM gene were determined by polymerase chain reaction. RESULTS: Tetracycline resistance was detected in 50.8% of the evaluated strains. The TRNG strains containing the tetM plasmid constituted 13.8% of all the evaluated strains. Dutch type tetM constituted 12.3% and American type tetM 1.5% of all the evaluated strains. In the remaining TRNG strains, resistance to tetracyclines was presumably chromosome-encoded. The minimal inhibitory concentration (MIC) of tetracycline ranged from 0.25 to 32.0 mg/l, MIC50 = 2.0 mg/l, MIC90 = 32.0 mg/l. The MIC of doxycycline ranged from 0.25 to 32.0 mg/l, MIC50 = 4.0 mg/l, MIC90 = 16.0 mg/l. CONCLUSIONS: Unlike most of European countries, in 2012-2013 in Poland, the Dutch type tetM was found to be much more common than the American type. Minimal inhibitory concentration values of tetracycline and doxycycline were similar, with doxycycline exhibiting a somewhat lower effectiveness in vitro than tetracycline towards chromosome-mediated tetracycline resistant strains of N. gonorrhoeae.

Dominating types of penicillinase-plasmids in Neisseria gonorrhoeae strains isolated in 2010-2012 in Warsaw.

INTRODUCTION: The reason of Neisseria gonorrhoeae resistance to penicillin is often production of TEM beta-lactamases encoded by plasmids. The most common types of the plasmid are Africa, Asia and Toronto/Rio. Another reason of resistance can be mutations in bacterial chromosome. The aim of the study was to investigate the types of plasmids occurring in in Neisseria gonorrhoeae strains isolated in 2010-2012 in Warsaw. MATERIAL AND METHODS: From 218 isolated in 2010, 2011 and at the beginning of 2012 from patients of Medical University in Warsaw we selected 12 strains producing beta- lactamase (penicillinase producing N. gonorrhoeae, PPNG). d B-tests to investigate bacterial sensitivity to penicillin and cefiriaxon. The types of plasmids were determined with PCR. RESULTS: The Beta-lactamases were encoded by Toronto/Rio (41,7%), Asia (33,3%) and Africa (25,0%) plasmids. All the strains were resistant to penicillin (MIC 2-8 mg/L) and sensitive to ceftriaxon (MIC 0,004-0,032 mg/L). CONCLUSIONS: All of the investigate PPNG strains were penicillin resistant and ceftriaxon sensitive. The dominating type of the penicillinase plasmid was Toronto/Rio.

Susceptibility to ceftriaxone and occurrence of penicillinase plasmids in Neisseria gonorrhoeae strains isolated in Poland in 2012-2013.

Recent years have seen rising concerns over increasing antibiotic resistance of the gonorrhea-causing bacterium, Neisseria gonorrhoeae. This is especially true for third-generation cephalosporins, which are currently recommended for the treatment of such infections. Therefore, susceptibility to these antibiotics should be monitored internationally to the greatest extent possible. The susceptibility of N. gonorrhoeae strains to ceftriaxone and penicillin, as well as production of beta-lactamase by the Cefinase test was determined. Moreover, the presence and type of penicillinase plasmids were determined by PCR. All strains were susceptible to ceftriaxone, the minimal inhibitory concentration (MIC) values ranged from 0.002 to 0.125 mg/L; MIC50 was =0.016 mg/L and MIC90 was =0.064 mg/L. As much as 7.7 % of the strains demonstrated ceftriaxone MIC of 0.125 mg/L. For penicillin, the MICs ranged from 0.064 to 32 mg/L; MIC50 was =0.5 mg/L and MIC90 was =4 mg/L. It was shown that only 1.5 % of the strains were sensitive to penicillin according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Among the penicillin-resistant strains, six (30.0 %) produced penicillinase. The MICs of penicillin were substantially higher for penicillinase-producing than for penicillin-resistant, penicillinase-negative strains. MICs of ceftriaxone for penicillinase-producing strains were low (0.002-0.016 mg/L). Three of the penicillinase-producing strains possessed plasmids of African type (50 %) and three Toronto/Rio type (50 %). An increase of the proportion of beta-lactamase-positive strains in the last years as well as emergence of strains with elevated MIC of ceftriaxone indicate a need to constantly monitor N. gonorrhoeae strains for their susceptibility to beta-lactam antibiotics, as well as for their ability to produce beta-lactamases.

[Susceptibility of Neisseria gonorrhoeae strains isolated in Poland in 2012-2013 to spectinomycin]. / Wrazliwosc na spektynomycyne szczepów Neisseria gonorrheae izolowanych w Polsce w latach 2012-2013.

INTRODUCTION: A rapid growth of the antibiotic resistance among Neisseria gonorrhoeae strains was recently observed in many countries. The common resistance or decreased susceptibility to penicillin, tetracyclines, ciprofloxacin, azithromycin as well as emergence of the first strains resistant to ceftriakson and cefixim is a cause of an anxiety worldwide. Spectinomycin may constitute an alternative therapy of gonorrhoea except of pharyngeal infection. METHODS: The susceptibility to spectinomycin of 65 N. gonorrhoeae strains isolated in the second half of 2012 and the first half of 2013 in Dermatology and Venereology Clinic in Warsaw was investigated. The E-Tests (bioMerieux) were performed and the results (sensitive or resistant) were interpreted according to EUCAST and CLSI recommendations. RESULTS: The MIC (Minimal inhibitory concentration) values of spectinomycin for the investigated strains ranged from 4,0 mg/L to 32 mg/L, MIC50=16,0 mg/L and MIC=90=16,0 mg/L. It was shown that 100% of the strains was sensitive to spectinomycin according to EUCAST as well CLSI tables. CONCLUSIONS: High susceptibility of the investigated strains to spectinomycin suggests that the drug can be used in the therapy of the resistant gonorrhoea in monotherapy or combined with other drugs.

[Resistance to ciprofloxacin of Neisseria gonorrhoeae strains isolated in Poland in 2012-2013]. / Opornosc na ciprofloksacyne szczepów Neisseria gonorrhoeae izolowanych w Polsce w latach 2012-2013.

INTRODUCTION: Ciprofloxacin is commonly used in Poland specially for the treatment of urinary tract infections including urethritis. Patients are often treated without pathogen identification and antimicrobial resistance tests. Neisseria gonorrhoeae infection is one of the most common causes of urethritis in Poland. The resistance of bacteria to a wide range of antibiotics including ciprofloxacine makes the therapy of gonorrhoea more difficult. The mechanism of ciprofloxacine action depends on inactivation of bacterial topoisomerase II (gyrase) and topoisomerase IV. A resistance to ciprofloxacine occurring in Neisseria gonorrhoeae is mainly due to mutations in bacterial gyrA (encoding topoisomerase II) and/or parC (encoding topoisomerase IV ) genes. High level resistance is an effect of combination of three or four mutations. Another, less important mechanism of ciprofloxacin resistance, that can coexist with mutations in gyrA and parC genes related to the overproduction of membrane pumps proteins. MATERIAL AND METHODS: 65 Neisseria gonorrhoeae strains isolated from patients of Department of Dermatology and Wenereology in Warsaw in the second half of 2012 and first of 2013 was investigated. The strains were cultured on chocolate agar plates in a 5% CO2 atmosphere at 37 degrees C and identified by colony morphology, Gram stain and oxidase reaction, followed by carbohydrate utilization test. Ciprofloxacin susceptibility was determined by E-Tests (bioMerieux). Bacteria were incubated at 35 degrees C in 5% CO2 for 24 h on chocolate agar plates. Tests were performed according to producers recommendations. The results (sensitive or resistant) were interpreted according to EUCAST recommendations. RESULTS: The MIC (Minimal inhibitory concentration) of Ciprofloxacin in investigated strains ranged from 0,002 to > 32 mg/L, MIC50 = 8 mg/L, MIC90 = > 32 mg/L. It was shown that only 38.5% of the strains were sensitive to ciprofloxacin according to EUCAST criteria from 2013 year. CONCLUSIONS: Due to the high percentage of ciprofloxacin resistant Neisseria gonorrhoeae strains (more than 61%) the antibiotic should not be used for the treatment of gonorrhoea in Poland.

[Resistance to azithromycin of Neisseria gonorrhoeae strains isolated in Poland in 2012-2013 years]. / Opornosc na azytromycyne szczepów Neisseria gonorrhoeae izolowanych w Polsce w latach 2012-2013.

INTRODUCTION: Azithromycin is one of the most commonly used macrolide antibiotics. As other macrolides it inhibits bacterial proteins synthesis by binding with V domene of bacterial 23S rRNA. Resistance to azithromycin can be related to: 1. Mutations in gene encoding 23S rRNA. Significant effect on azithromycin MIC (minimal inhibitory concentration) is not observed when the mutation occurs only in 1 allele. In case of mutations occurring in 4 alleles, more common mutation C2611T is associated with MIC 2-16 mg/L and the second mutation A2059T results in high level resistance to azithromycin MIC > 256 mg/l 2. Over- production of membrane pumps proteins MtrCDE and MacAB, that remove antibiotics from bacterial cells. The mechanism is not able to cause azithromycin resistance itself but coexisting with other mechanisms of resistance can additionally increase MIC. 3. Synthesis of 23S rRNA methylases ErmB, ErmF, ErmC, ErmA. These enzymes cause demethylation of adenine (A2058) in V domain of 23S rRNA. The mechanism was common in the past, but it has been replaced by mutations in in V domain of 23S rRNA. Nowadays 23S rRNA methylases are very rare in N. gonorhoeae. METHODS: Sixty five Neisseria gonorrhoeae strains isolated from patients of the Department of Dermatology and Venereology in Warsaw in the second half of 2012 and first of 2013 were investigated. The strains were cultured on chocolate agar plates in a 5% CO2 atmosphere at 37 °C and identified by colony morphology, Gram staining and oxidase reaction, followed by carbohydrate utilization test. Azithromycin susceptibility was determined by E-Tests (bioMerieux). Bacteria were incubated at 37°C in 5% CO2 for 24 h on chocolate agar plates. Tests were performed according to producers recommendations. The results (sensitive or resistant) were interpreted according to EUCAST recommendations. RESULTS: The MIC of azithromycin in investigated strains ranged from 0,064 to 4 mg/L, MIC50 = 0.5 mg/L, MIC90 = 2 mg/L. It was shown that only 38.5% of the strains were sensitive to azithromycin according to EUCAST criteria from 2014 year and 89.3% of the strains were sensitive to azithromycin according to CDC criteria from 2014 year. CONCLUSIONS: The percentage of azithromycin resistant Neisseria gonorrhoeae strains is increasing in Poland and the antibiotic should not be used in monotherapy as gonorrhoea patients. It should only be used in combination with ceftriaxone or cefixime.