Investigation of antibiotic resistance determinants and virulence factors of uropathogenic Escherichia coli.

Multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) are prevalent throughout the world resulting in a major public health burden. In this research, we isolated and identified 28 MDR UPEC from one university hospital in China, investigated MDR and pathogenic mechanisms by PCR, including 55 antibiotic resistance determinants (ARDs) genes, 13 genetic markers of mobile genetic elements (MGEs) and 6 virulence factors (VFs) genes. In these isolates, we identified 23 ARDs genes and 6 genetic markers of MGEs that played a key role in MDR phenotypes. In addition, we found 2 VFs genes, hofQ and ompT, which could be associated with pathogenicity and invasiveness of these strains in urinary tract infections (UTIs).

Draft Genome Sequence of a Klebsiella pneumoniae Strain (New Sequence Type 2357) Carrying Tn3926.

We present the draft genome sequence of a Klebsiella pneumoniae carbapenemase-producing sequence type 2357 (ST2357) strain, NB60, which contains drug-resistant genes encoding resistance to beta-lactams, fluoroquinolones, aminoglycosides, trimethoprim-sulfamethoxazole, colistin, macrolides, and tetracycline. Strain NB60 was isolated from human blood, making it an important tool for studying K. pneumoniae pathogenesis.

Draft Genome Sequence of Uropathogenic Escherichia coli Strain NB8.

Escherichia coli NB8 is a clinical pyelonephritis isolate. Here, we report the draft genome sequence of uropathogenic E. coli NB8, which contains drug resistance genes encoding resistance to beta-lactams, aminoglycosides, quinolones, macrolides, colistin, sulfonamide-trimethoprim, and tetracycline. NB8 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis.

[Clinical features and molecular characteristics of methicillin-resistant Staphylococcus aureus in children].

OBJECTIVE: To study the clinical and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) infection in children. METHOD: A total of 37 MRSA strains were isolated from hospitalized patients in Children's Hospital of Fudan University from March 2009 to November 2011. The clinical characteristics were investigated by a cohort study. Furthermore, the mecA, Panton-Valentine leucocidin (PVL) genes were detected by polymerase chain reaction (PCR), and the genotypes of SCCmec were determined by multiplex PCR. RESULT: (1) Among the 37 MRSA isolates, infections with 21 were acquired from hospital (HA-MRSA), and 16 isolates were acquired from community (CA-MRSA). (2) In the study, MRSA frequently caused respiratory tract infection, and most of the strains were isolated from intensive care unit (ICU). (3) CA-MRSA was most frequently associated with skin and soft tissue infections (SSTI), suppurative tonsillitis, even pneumonia and septicemia. HA-MRSA infection was more aggressive, most frequently associated with pneumonia, septicemia, and central nervous system (CNS) infections, such as meningitis. In children with fever caused by HA-MRSA or CA-MRSA infection, HA-MRSA showed a longer duration of fever, for 10.5 days. C-reactive protein (CRP) level caused by HA-MRSA (63.00 mg/L) was higher than CA-MRSA (9.50 mg/L) , and there were statistically significant differences between the groups (t = 2.5670, P < 0.05). However, there were no statistically significant differences between the groups in white blood cell count (WBC) or procalcitonin (PCT) level. (4) Among 37 MRSA isolates, the whole isolates were mecA gene positive (100%). SCCmec genotyping results showed that the most frequent SCCmec types were type III, 17 isolates, the others including type IV 8 isolates, type II1 isolates, nontypable 11 isolates, type I and type V were not found in this group. Therein, among 21 HA-MRSA isolates, SCCmec III was the most common, 15 isolates, type IV 1 isolates, nontypable 5 isolates; among 16 CA-MRSA isolates, SCCmec type IV was the most common, 7 isolates, type III 2 isolates, type II 1 isolate, nontypable 6 isolates. (5) Among the 37 MRSA isolates, 28 were PVL gene positive; and among 21 HA-MRSA isolates, 17 were PVL gene positive; Among 16 CA-MRSA isolates, 11 were PVL gene positive; There were no statistically significant differences between the groups (χ(2) = 0.735, P > 0.05) . CONCLUSION: Compared with CA-MRSA, HA-MRSA infection was more aggressive, and induced higher C reactive protein; the dominant epidemic strains of CA-MRSA was SCCmec type IV, and HA-MRSA was SCCmec type III; the positive rate of PVL gene was high.

[Relationship between drug resistance of Pseudomonas aeruginosa isolated from burn wounds and its mobile genetic elements].

OBJECTIVE: To investigate the relationship between the drug resistance of Pseudomonas aeruginosa (PA) isolated from burn patients wounds and its mobile genetic elements, including plasmid, transposon, and integron. METHODS: Thirty-two strains of PA were isolated from wounds exudate of hospitalized burn patients in Ningbo No. 2 Hospital. PA drug sensitivity was determined using GNS-448 drug sensitivity card and K-B tests. The genetic markers of plasmid, transposon and integron including traA, traF, tnpA, tnpU, merA, int I 1 were amplified by PCR and verified by gene sequencing. RESULTS: Drug resistant rate of 32 PA strains to gentamicin, amikacin, cefoperazone/sulbactam, ciprofloxacin was 43.7%, 32.0%, 46.8%, 49.9%, respectively. PA drug resistant rates to piperacillin, cefotaxime, ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, levofloxacin, imipenem and meropenem were all above 56.0%. Seventeen out of 32 PA strains were found to carry transposon and (or) integron genetic markers. One strain was positive for both tnpA and merA, 8 strains were positive for both merA and int I 1, 1 strain was only positive for tnpA, 2 strains were only positive for merA, and 5 strains were positive for int I 1 only. CONCLUSIONS: PA isolated from burn wounds of hospitalized patients in Ningbo No. 2 Hospital is seriously drug resistant, which may relate with its high positive rate of mobile genetic elements of transposon and (or) integron.

Drug-resistant gene based genotyping for Acinetobacter baumannii in tracing epidemiological events and for clinical treatment within nosocomial settings.

BACKGROUND: Acinetobacter baumannii has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. However, of the frequently used methods, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) in Acinetobacter baumannii genotyping lack the direct molecular proof of drug resistance. This study was conducted to establish a typing method based on drug resistant gene identification in contrast to traditional PFGE and AFLP in the period of nosocomial epidemic or outbreak. METHODS: From January 2005 to October 2005, twenty-seven strains of Acinetobacter species from Intensive Care Units, the Second Affiliated Hospital in Ningbo were isolated, including both epidemic and sporadic events. Susceptibility test, PFGE, AFLP and drug resistance gene typing (DRGT) were carried out to confirm the drug resistance and analyze the genotyping, respectively. PFGE was used as a reference to evaluate the typeability of DRGT and AFLP. RESULTS: Twenty-seven strains of Acinetobacter displayed multiple antibiotic resistance and drug resistant genes, and beta-lactamase genes were detected in 85.2% strains. The result of DRGT was comparable to PFGE in Acinetobacter strains with different drug resistance though a little difference existed, and even suggested a molecular evolution course of different drug-resistant strains. AFLP showed great polymorphism between strains and had weak ability in distinguishing the drug resistance. CONCLUSION: Compared to AFLP and PFGE, DRGT is useful to analyze localized molecular epidemiology of nosocomial infections and outbreaks, which would benefit clinical diagnosis and therapy.

[Application of nested PCR and sequencing technique to detect point mutations of the 23S rRNA gene of Mycoplasma pneumoniae].

OBJECTIVE: To establish a quick method to detect drug resistance of Mycoplasma pneumoniae (MP) and study the condition of drug resistance in MP infection. METHODS: MP 23S rRNA target gene in throat swab specimens from 200 patients with suspected MP infection was detected by using nested PCR and DNA sequencing. The result of 23S rRNA gene detection was confirmed by MP isolation and drug susceptibility test in vitro for reliability. RESULTS: Of the 200 clinical specimens, 64 were proved to be positive for MP through MP-IgM antibody, MP specific 16S rRNA nested PCR and MP isolation . The 23S rRNA gene was amplified and the gene sequence was compared with MP reference strain in Genbank, 26 were identical to the reference strain, 38 had a point mutation in 23S rRNA. Among them, 35 had A to G mutation at position 2063, 1 had A to C mutation at position 2063 and 2 had A to G mutation at position 2064, the percentage of drug resistance was 59.4%. The sensitivity of the gene detection method was 10(2) ccu/ml and it was confirmed to be reliable by MP isolation and drug susceptibility test. CONCLUSIONS: The gene detection method could detect MP drug resistant gene directly from clinical specimen, which has the advantages of high specificity, high sensitivity and quickness. It is of great significance for diagnosis of MP infection because MP isolation is difficult and time-consuming.

[Analysis on 168 rRNA methylase genes and aminoglycoside modifying enzymes genes in Enterobacter cloacae in China].

OBJECTIVE: To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes (AMEs) genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital, Huzhou district, Zhejiang province, China. METHODS: 40 strains of Enterobacter cloacae were isolated from the inpatients between September, 2003 and November, 2004. 5 kinds of 16S rRNA methylase gene (including armA, rmtA, rmtB, rmtC and rmtD) and 9 kinds of AMEs gene [including aac (3)-I, aac(3)-II, aac(3)-III, aac(3)-IV, aac(6')-Ib, aac(6')-II, ant(3")-I, ant(2")-I and aph(3')-VI] were analyzed by PCR and verificated by DNA sequencing. RESULTS: In 40 strains of Enterobacter cloacae, the positive rates of genes of rmtB, aac(3)-II, aac(6')-I b, ant(3")-I, ant(2")-I and aph(3')-VI were 12.5% (5/40), 27.5% (11/40), 72.5% (29/40), 32.5% (13/40), 5.0% (2/40) and 5.0% (2/40), respectively. 8 kinds of the rest of genes were all tested negative. The total positive rate of AMEs gene was 85.0% (34/40). Among 29 strains of Enterobacter cloacae that the aac (6')-I b gene was positive, through PCR and verification by DNA sequencing, 7 strains (24.1%) were confirmed to take the aac(6')-I b-cr (the GenBank register number: EF375620, EU159121) alone, 18 strains (62.1%) were confirmed to take the aac(6')-I b-Suzhou (EU085533) alone, 3 strains (10.3%) were confirmed to take both aac(6')-I b-Suzhou and aac(6')-I b-cr while only 1 (3.4%) was aac(6')-I b (the classical type). CONCLUSION: There was lower positive rate of 16S rRNA methylase gene but very high AMEs genotypes in Enterobacter cloacae isolated from inpatients and the finding of rmtB gene was reported for the first time in the world. At least 5 kinds of AMEs gene existed in Enterobacter cloacae were isolated and they were the new host of both gene of aac(6')-I b- cr and aac(6')-I b-Suzhou, with aac(6')-I b-Suzhou gene was the predominance subtype in aac(6')-I b.

[Emergence of 16S rRNA methylase gene rmtB in Klebsiella pneumoniae isolates from the inpatients in Zhejiang Province, China].

OBJECTIVE: To investigate the presence and genetic background of 16S rRNA methylase gene and Aminoglycoside modifying enzymes (AMEs) genes in Klebsiella pneumoniae isolated from the People's Liberation Army 98th Hospital, Huzhou district, Zhejiang province, China. METHODS: 25 strains of Klebsiella pneumoniae were isolated from the inpatients between September, 2005 and April, 2006. 6 kinds of 16S rRNA methylase gene (including armA, rmtA, rmtB, rmtC, rmtD and npmA), 6 kinds of AMEs genes [including aac (3)-I, aac (3)-II, aac (6')-I, aac (6')-II, ant (3")-I and ant (2")-I], intI1, intI2, intI3, mercuric reductase gene merA (merA gene were the collective genetic markers of transposons of Tn21 and Tn501) and tnpA (tnpA gene were the collective genetic markers of transposons of Tn1, Tn2,Tn3 and Tn1000) were analyzed by PCR and verificated by DNA sequencing. RESULTS: In 25 strains of Klebsiella pneumoniae, the positive rate of genes of rmtB, aac (3)-II, aac (6')-I, ant (3")-I and intI1 were 60.0% (15/25), 4.0% (1/25), 48.0% (12/25), 60.0% (15/25) and 96.0% (24/25), respectively. The rest 12 kinds of genes were all tested negative. The total positive rate of 6 kinds of AMEs gene was 84.0% (21/25). CONCLUSION: There were very high positive rate on both genes of rmtB and AMEs genotypes in Klebsiella pneumoniae isolated from inpatients, and this was the first report of the emergence of 16S rRNA methylase gene rmtB in Klebsiella pneumoniae identified in Zhejiang province, China.

[Identification of a new subtype of blaADC produced by Acinetobacter baumannii isolated in children].

OBJECTIVE: To investigate the genotype of blaADC which was a kind of AmpC produced by Acinetobacter baumannii (AB), isolated through the detection of 28 similar strains among children. METHODS: 28 strains of AB were collected and isolated from the Pediatrics clinic during 2006, and were identified through bacteria and susceptibility test using Vitex-32 automicroscan GNI and GNS cards. The genotype of blaADC was confirmed by polymerase chain reaction (PCR) and them sequenced. RESULTS: 3 of the 28 strains of AB showed multi-drugs resistance, with a positive rate of 10.71%. blaADC was discovered in 17 of the 28 strains and the positive rate was 60.71%. All the 28 strains of AB were resistant to Cefoxitin. blaADC positive strains were all sensitive to Ampicil/Sulbactam, and only one of them was resistant to Piperacillin/Tazobactan. There were no blaADC genes discovered in the strains that were resistant to Ampicil/Sulbactam or Piperacillin/Tazobactan. There were changes of amino acids on the site 4, 242, 342 and 376 in the sequence of blaADC of No.2 strain, comparing to gi /7258342/ emb /CAB77444. 1/ in GenBank. CONCLUSION: Above 60% of the AB isolated in children were carrying blaADC while a strain was collected from them at random. When they were undertaken nucleotide sequence analysis, significant difference was found from the others that landed in GenBank, which identified itself as new subtype.

[Study on fluoroquinolone resistance and the relationship between resistance and mutations of gyrA and parC in Neisseria gonorrhoeae].

OBJECTIVE: To study the phenotypic and genotypic resistance to Fluoroquinolones in Neisseria gonorrhoeae (NG) isolated in Jiangsu province of China. METHODS: In-vitro, susceptibility testing of ciprofloxacin and levofloxacin against ninety-five clinical isolates were determined by agar dilution method. Detection of mutation in the gyrA and parC genes was performed by polymerase chain reaction (PCR) assay and sequence analysis. RESULTS: The clinical isolates demonstrated 100% resistance to ciprofloxacin. Based on gyrA and parC mutations, 18 types could be categorized among the 54 isolates. Based on the same gyrA mutations,isolates with high MIC appeared to have had more mutations in parC gene. CONCLUSION: The status of resistance to ciprofloxacin in NG was quite serious, and ciprofloxacin treatment for the treatment of NG infections in Jiangsu province should not be recommended. The results from this study suggested that mutations in the parC gene had contributed to the development of high Fluoroquinolone resistance in NG.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Jiangsu Province, China, with a focus on fluoroquinolone resistance.

In this study, the phenotypic and genotypic resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Jiangsu Province, China, was analysed. In vitro susceptibility testing of eight antimicrobial agents, including ciprofloxacin and levofloxacin, against 95 clinical isolates was carried out. Detection of mutations in the gyrA and parC genes was performed by sequence analysis. The clinical isolates demonstrated 100% resistance to ciprofloxacin and 98.9% non-susceptibility to levofloxacin. All of the isolates were susceptible to cefotaxime and ceftriaxone. For cefepime, spectinomycin and tetracycline, 98.9, 94.7 and 1.1% of the isolates were susceptible, respectively. None of the isolates was susceptible to penicillin. Five types based on gyrA mutations could be categorized among 54 isolates with seven different mutation sites found on their parC gene. Analysis of sequence results showed that the gyrA mutation Asp-95-->Ala and the parC mutations Ser-87-->Arg and Ser-87-->Asn made a significant contribution to the resistance to fluoroquinolones, in addition to double mutations found in each gene. Therefore, the use of fluoroquinolones in the treatment of N. gonorrhoeae infections in Jiangsu Province is not recommended, while the use of third- and fourth-generation cephalosporins and spectinomycin is recommended.

[Study on the molecule epidemiological between resistances of 7 genes interrelated 4 antibiotic to isolated Streptococcus pneumoniae in children].

OBJECTIVE: To investigate the molecule epidemic for 7 genes interrelated penicillin, erythromycin, tetracycline, vancomycin resistance of isolated Streptococcus pneumoniae (SP) in children at Suzhou area. METHODS: (1) Thirty-one pneumococcal isolates were collected from respiratory tract secretions of children with respiratory diseases from Nov 2002 to Apr 2003 at the Children's Hospital of Suzhou University (reference strain ATCC49619). (2) Penicillin susceptibility was determined by E-test, while erythromycin, tetracycline, vancomycin were determined by K-B disk. (3) The detecting of pbp2B, ermA/B, mefA, tetM, vanA, vanB genes by PCR, Sequencing pbp2B genes, Contrasting pbp2B DNA sequences among pneumococcal isolates and SP R6 [penicillin sensitive (www.ncbi.nlm.gov/nucleotide, NC-003098)]. RESULTS: Of thirty-one isolates studied, the results were shown as follows; (1) Penicillin sensibility 38.7% (n = 12), penicillin resistance 61.3% (n = 19), pbp2B mutation 64.5% (n = 20); (2) Erythromycin sensibility 9.7% (n = 3), erythromycin resistance 90.3% (n = 28), ermA/B 71% (n = 22), mefA 32.1% (n = 10), ermA/B + mefA 87.1% (n = 27); (3) Tetracycline sensibility 9.7% (n = 3), tetracycline resistance 90.3% (n = 28), tetM 90.3% (n = 28); (4) Vancomycin sensibility 100% (n = 31), vanA, vanB all 0%. CONCLUSION: Among pneumococcal isolates at our area, penicillin, erythromycin, tetracycline resistance were high, vancomycin was sensitive. Detecting 7 genes interrelated penicillin, erythromycin, tetracycline, vancomycin resistance expressed genotypies for antibiotic resistances in pneumococcal isolates.

[Relation of pbp2B, ermB, ermA/B, mefA genes with resistance to penicillin and erythromycin among Streptococcus pneumoniae isolates from children].

OBJECTIVE: To investigate the relation of pbp2B, ermB, ermA/B and mefA genes to penicillin and erythromycin resistance among isolated Streptococcus pneumoniae (Sp) in children. METHODS: Twenty-six strains of Sp were collected from September 2002 to April 2003 at the Children Hospital of Suzhou University. (1) Twenty-six pneumococcal isolates were obtained from respiratory tract secretions of children with respiratory diseases. (2) Susceptibility of the isolates to penicillin, cefuroxime, ceftriaxone, cefotaxime and erythromycin was determined by E-test. (3) The genes pbp2B, ermB, ermA/B and mefA of the isolates were detected with PCR. (4) The PCR product of pbp2B gene was sequenced. (5) DNA sequences of pbp2B of pneumococcal isolates were compared with those of SpR6 [penicillin sensitive (www.ncbi.nlm.gov/nucleotide, NC-003098)]. RESULTS: Among the 26 isolates studied, pbp2B gene mutation was found in 15(58%) isolates, all were point mutation of A, B, C and D genotypes which were seen in 11(73%), 2(13%), 1(7%) and 1(7%), respectively. The numbers of isolates susceptible to penicillin, cefuroxime, ceftriaxone and cefotaxime were 9(82%), 10(91%), 11(100%) and 11(100%), of 11 non-mutation isolates;numbers of isolates resistant to penicillin, cefuroxime, ceftriaxone, and cefotaxime were 13(87%), 11(73%), 1(7%) and 1(7%) out of 15 isolates with mutation.ErmB, ermA/B, mefA and erm/mef genes were positive in 9(35%), 16(62%), 7(27%) and 21(81%)isolates. MIC of erythromycin was 2 to > 256 mg/L among pneumococcal isolates with erm/mef genes. CONCLUSION: Among antibiotic resistant pneumococcal isolates in the area, the main basis of penicillin resistance was the mutation of pbp2B genes. Genotype A mutation had the highest rate among the isolates with mutation and manifested as resistance to penicillin and cefuroxime. Expression of either all or any of the ermA, ermB and mef genes led to erythromycin resistance. Antibiotics resistant Sp strains in this area are forming a challenge to efficacy of penicillin and erythromycin.

Relationship between antimicrobial resistance and aminoglycoside-modifying enzyme gene expressions in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by multi-drug resistance Acinetobacter baumannii is very difficult to treat. This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycoside-modifying enzymes including N-acetyltransferases and O-phosphotransferases in Acinetobacter baumannii. METHODS: Bacterial identification and antimicrobial susceptibility test were performed by Phoenix system in 247 strains of Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multi-drug resistant Acinetobacter baumannii were detected by agar dilution. Four aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer. RESULTS: The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50%. Imipenem and meropenem showed high antibacterial activities with resistance rates of 3.2% and 4.1%. MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multi-drug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 93.3%, respectively. But their resistance rates to tobramycin, netilmicin and neomycin were 86.7%, 93.3% and 46.7%, respectively. Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected. Their positive rates were 93.3%, 20.0% and 20.0%, respectively. These three genes existed simultaneously in No.19 strain. Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 97.9% and 99.7% identities with GenBank genes (AY307113, S68058 and AY307114). CONCLUSION: Multi-drug resistant Acinetobacter baumannii strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.