Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae.

Objective: In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression. Methods: We created the ampG gene deletion mutant strains Kp1-ΔampG and Kp NTUH-K2044-ΔampG with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-ΔampG-RC drug resistance model strains with plasmid pACYC184. We constructed the ampG knock-in strains by introducing the ampG genes of Kp1, Enterobacter cloacae 029M, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC25922, and Salmonella typhimurium LT2 into the ampG gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of ampC and ampG mRNAs. Results: Compared with Kp1, the induction phenotype of the ampC of Kp1-ΔampG strain disappeared, the ampC expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 µg/mL to 1 µg/mL. Based on Kp1, five strain were successfully constructed to complement the ampG genes from five knock-in strain, and all of the above complemented strains showed inducible expression of ampC and restored the expression of ampG to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime (P < 0.05). The ampC and ampG genes were barely expressed in Kp NTUH-K2044-ΔampG-RC when compared with Kp NTUH-K2044-RC. The expressions of ampG and ampC in each knock-in strain were recovered, the induction phenotype of ampC was restored, and the MIC values of cefoxitin and ceftazidime were increased. (P < 0.05). Conclusion: In this study, we found that ampG was an essential regulator for the plasmid-mediated ampC-induced expression in K. pneumoniae.

E. coli diversity: low in colorectal cancer.

BACKGROUND: Escherichia coli are mostly commensals but also contain pathogenic lineages. It is largely unclear whether the commensal E. coli as the potential origins of pathogenic lineages may consist of monophyletic or polyphyletic populations, elucidation of which is expected to lead to novel insights into the associations of E. coli diversity with human health and diseases. METHODS: Using genomic sequencing and pulsed field gel electrophoresis (PFGE) techniques, we analyzed E. coli from the intestinal microbiota of three groups of healthy individuals, including preschool children, university students, and seniors of a longevity village, as well as colorectal cancer (CRC) patients, to probe the commensal E. coli populations for their diversity. RESULTS: We delineated the 2280 fresh E. coli isolates from 185 subjects into distinct genome types (genotypes) by PFGE. The genomic diversity of the sampled E. coli populations was so high that a given subject may have multiple genotypes of E. coli, with the general diversity within a host going up from preschool children through university students to seniors. Compared to the healthy subjects, the CRC patients had the lowest diversity level among their E. coli isolates. Notably, E. coli isolates from CRC patients could suppress the growth of E. coli bacteria isolated from healthy controls under nutrient-limited culture conditions. CONCLUSIONS: The coexistence of multiple E. coli lineages in a host may help create and maintain a microbial environment that is beneficial to the host. As such, the low diversity of E. coli bacteria may be associated with unhealthy microenvironment in the intestine and hence facilitate the pathogenesis of diseases such as CRC.

A rapid loop-mediated isothermal amplification (LAMP) method for detection of the macrolide-streptogramin type B resistance gene msrA in Staphylococcus aureus.

Macrolide-streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25min at an isothermal temperature of 62°C. More importantly, the msrA gene could be detected at a sensitivity as low as 100pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene.

DHA-1 plasmid-mediated AmpC ß-lactamase expression and regulation of Klebsiella pnuemoniae isolates.

The present study aimed to investigate the regulatory mechanism of the AmpC enzyme by analyzing the construction and function of AmpCR, AmpE and AmpG genes in the Dhahran (DHA)­1 plasmid of Klebsiella pneumoniae (K. pneumoniae). The production of AmpC and extended­spectrum ß­lactamase (ESBL) were determined following the cefoxitin (FOX) inducing test for AmpC, preliminary screening and confirmation tests for ESBL in 10 DHA­1 plasmid AmpC enzymes of K. pneumoniae strains. AmpCR, AmpD, AmpE and AmpG sequences were analyzed by polymerase chain reaction. The pACYC184­X plasmid analysis system was established and examined by regulating the pAmpC enzyme expression. The electrophoretic bands of AmpCR, AmpD, AmpE and AmpG were expressed. Numerous mutations in AmpC + AmpR (AmpCR) and in the intergenic region cistron of AmpC­AmpR, AmpD, AmpE and AmpG were observed. The homology of AmpC and AmpR, in relation to the Morganella morganii strain, was 99%, which was determined by comparing the gene sequences of Kp1 with those of Kp17 AmpCR. The specific combination of AmpR and labeled probe demonstrated a band retarded phenomenon and established a spatial model of AmpR. All the enzyme production strains demonstrated Val93→Ala in AmpG; six transmembrane domains were found in AmpE in all strains, with the exception of Kp1 and Kp4, which had only three transmembrane segments that were caused by mutation. The DHA­1 plasmid AmpC enzymes encoded by plasmid are similar to the inducible chromosomal AmpC enzymes, which are also regulated by AmpD, AmpE, AmpR and AmpG.

Characteristics of qacA/B-positive Staphylococcus aureus isolated from patients and a hospital environment in China.

OBJECTIVES: This study was designed to demonstrate the characteristics of qacA/B-positive Staphylococcus aureus in China. METHODS: One hundred and forty-five MRSA and 178 MSSA from clinical specimens from seven hospitals in different regions of China, 70 MRSA from superficial sites of patients and 106 MRSA from environmental samples from an ICU were collected and screened for the presence of the qacA/B gene. The qacA/B-positive isolates and 72 randomly selected qacA/B-negative control isolates were further characterized by MLST, spa typing and detection of toxin genes, as well as antimicrobial and chlorhexidine susceptibility. SCCmec typing was conducted for MRSA. PFGE was conducted for qacA/B-positive isolates. RESULTS: Twenty-five (7.8%) of the 321 MRSA isolates harboured qacA/B, including 11 isolates from clinical specimens (7.6%), 12 isolates from patients' superficial sites (17.1%) and 2 isolates from an ICU environment (1.9%). Ten and five qacA/B-positive MRSA were identified as ST239-t030-MRSA-III and ST239-t037-MRSA-III, respectively. Six PFGE clusters and five singletons were identified among the 25 qacA/B-positive MRSA. Only one (0.6%) of the 178 MSSA isolates harboured qacA/B. qacA/B carriage in MRSA was statistically associated with spa-t037 and the presence of mupA. Compared with qacA/B-negative MRSA, the qacA/B-positive MRSA exhibited a lower susceptibility to chlorhexidine and higher resistance rates to clindamycin and trimethoprim/sulfamethoxazole. CONCLUSIONS: Carriage of qacA/B, although it had a low prevalence, might be the main reason for declining susceptibility to chlorhexidine in MRSA from Chinese patients and is probably associated with spa-t037 and the presence of the mupA gene.

Characterization of Staphylococcus aureus from distinct geographic locations in China: an increasing prevalence of spa-t030 and SCCmec type III.

Staphylococcus aureus belongs to one of the most common bacteria causing healthcare and community associated infections in China, but their molecular characterization has not been well studied. From May 2011 to June 2012, a total of 322 non-duplicate S. aureus isolates were consecutively collected from seven tertiary care hospitals in seven cities with distinct geographical locations in China, including 171 methicillin sensitive S. aureus (MSSA) and 151 MRSA isolates. All isolates were characterized by spa typing. The presence of virulence genes was tested by PCR. MRSA were further characterized by SCCmec typing. Seventy four and 16 spa types were identified among 168 MSSA and 150 MRSA, respectively. One spa type t030 accounted for 80.1% of all MRSA isolates, which was higher than previously reported, while spa-t037 accounted for only 4.0% of all MRSA isolates. The first six spa types (t309, t189, t034, t377, t078 and t091) accounted for about one third of all MSSA isolates. 121 of 151 MRSA isolates (80.1%) were identified as SCCmec type III. pvl gene was found in 32 MSSA (18.7%) and 5 MRSA (3.3%) isolates, with ST22-MSSA-t309 as the most commonly identified strain. Compared with non-epidemic MRSA clones, epidemic MRSA clones (corresponding to ST239) exhibited a lower susceptibility to rifampin, ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole, a higher prevalence of sea gene and a lower prevalence of seb, sec, seg, sei and tst genes. The increasing prevalence of multidrug resistant spa-t030 MRSA represents a major public health problem in China.

Pseudomonas aeruginosa isolates of distinct sub-genotypes exhibit similar potential of antimicrobial resistance by drugs exposure.

Pseudomonas aeruginosa, a wide-spread opportunistic pathogen, often complicates clinical treatments due to its resistance to a large variety of antimicrobials, especially in immune compromised patients, occasionally leading to death. However, the resistance to antimicrobials varies greatly among the P. aeruginosa isolates, which raises a question on whether some sub-lineages of P. aeruginosa might have greater potential to develop antimicrobial resistance than others. To explore this question, we divided 160 P. aeruginosa isolates collected from cities of USA and China into distinct genotypes using I-CeuI, a special endonuclease that had previously been proven to reveal phylogenetic relationships among bacteria reliably due to the highly conserved 26-bp recognition sequence. We resolved 10 genotypes by I-CeuI analysis and further divided them into 82 sub-genotypes by endonuclease cleavage with SpeI. Eight of the 10 genotypes contained both multi-drug resistant (MDR) and less resistant isolates based on comparisons of their antimicrobial resistance profiles (ARPs). When the less resistant or susceptible isolates from different genotypes were exposed to eight individual antimicrobials, they showed similar potential to become resistant with minor exceptions. This is to our knowledge the first report to examine correlations between phylogenetic sub-lineages of P. aeruginosa and their potential to become resistant to antimicrobials. This study further alerts the importance and urgency of antimicrobial abuse control.

Identification of genotypes of plasmid-encoded AmpC beta-lactamases from clinical isolates and characterization of mutations in their promoter and attenuator regions.

We investigated the occurrence of AmpC beta-lactamases among Escherichia coli and Klebsiella pneumoniae isolates and determined the genotype of plasmid-mediated AmpC beta-lactamases at a medical center. The AmpC beta-lactamase promoter and attenuator were amplified from chromosomal DNA of high AmpC-producing E. coli isolates and sequenced. Antibiotic screening and 3D extract tests showed the presence of AmpC beta-lactamase in 3.56% of K. pneumoniae and 1.88% of E. coli isolates. Ten isolates (six K. pneumoniae and four E. coli) were positive for extended spectrum beta-lactamase (ESBL) as indicated by the double disc diffusion method. DHA-1 plasmid-encoded AmpC beta-lactamase was present in 10 K. pneumoniae isolates and four E.coli isolates. E. coli chromosomal AmpC beta-lactamase carried polymorphisms in the -42, -32, and -18 bases of the promoter and in the +26 and +27 bases of the attenuator, which may play a role in antibiotic resistance. The observed mutations may have clinical implications for the management of antibiotic-resistant infections.

The method of single-nucleotide variations detection using capillary electrophoresis and molecular beacons.

We demonstrate that single-nucleotide variations in a DNA sequence can be detected using capillary electrophoresis (CE) and molecular beacons (MBs). In this method, the region surrounding the site of a nucleotide variation was amplified in a polymerase chain reaction, then hybridize PCR products with each of MBs. The sequences of the PCR products are different at the site of 2,044 in exon of interleukin (IL)-13 which to be identified. Through denaturation, the PCR product became single strand and hybridized with the completely complementary MB. The MB-target duplexes were separated using CE and solution-based fluorescence techniques. The results show that in each reaction a fluorescent response was elicited from the molecular beacon which was perfectly complementary to the amplified DNA, but not from the other MB whose probe sequence mismatched the target sequence. The method of CE based on MBs is able to identify single-nucleotide variations in a DNA sequence and can discriminate the genotyping of the SNP between the homo- and heteroduplexes of DNA fragments.