Ethics statement
Ethical approval for this study was provided by the ethical review board of HTD in Ho Chi Minh City (HCMC), Vietnam.
Study setting and patient population
HTD is a 550-bed infectious disease hospital that serves as a primary and secondary facility for the surrounding local population in HCMC and a tertiary referral centre for infectious diseases for the 17 southern provinces of the country. Neonates, patients without infectious diseases, including those with surgical requirements, tuberculosis, cancer, primary haematological disorders or immunosuppression (other than HIV) are referred to other hospitals in the city.
Blood cultures were performed for patients in whom an infection was suspected on the basis of a fever (> 38 °C) or who had evidence of sepsis on the basis of the presence of two or more of the following features: fever (> 38 °C) or low temperature (< 36 °C); tachycardia (exact level according to age); tachypnea (exact level according to age); an elevated white cell count (> 12,000 cells/mm3) or depressed white cell count (< 4000 cells/mm3). There was no systematic change in the application of these criteria during the time course of the study. All data originating from consecutive patients admitted to the hospital who had a blood culture performed for suspected bloodstream infection between January 1st 2011 and December 31st 2013 were included in this retrospective study. Routinely, a member of the hospital staff recorded the date of blood draw, the patient’s age, sex, and suspected diagnosis, the number of blood culture bottles inoculated, the result of the culture (whether positive or negative) and the susceptibility of the isolate to commonly used antimicrobial agents. These are the source data for this study. For the purposes of these analyses blood samples taken for culture > 48 h after admission to HTD were classified as a Hospital Acquired Infection (HAI) and samples taken within 48 h of admission to HTD were classed as a Community Acquired Infection (CAI).
Blood culturing and bacterial identification
Venous blood cultures of 8–15 mL from adults and 2–5 mL of venous blood from infants and children were routinely obtained and inoculated into BACTECplus aerobic bottles (Becton Dickenson, USA). Inoculated BACTEC bottles were incubated at 37 °C in a BACTEC 9050 automated analyser for up to 5 days and sub-cultured when the machine indicated a positive signal. All sub-cultures were plated onto fresh sheep blood agar (Oxoid Unipath, Basingstoke, United Kingdom). Plates were incubated at 37 °C in air for 5 days and organisms were subsequently identified by standard methods including API20E and API20NE identification kits (Bio-Mérieux, France). Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were used as controls.
Antimicrobial susceptibility testing
The susceptibility to relevant antimicrobial agents was determined by the modified Bauer-Kirby disc diffusion method. Enterobacteriaceae were tested with discs containing chloramphenicol (30 μg), ampicillin (10 μg), co-trimoxazole (trimethoprim 1.25 μg/sulfamethoxazole 23.75 μg), ceftriaxone (30 μg), ofloxacin (5 μg) and gentamicin (10 μg). The breakpoint zone sizes were interpreted according to CLSI guidelines [11].
The double disk diffusion method [12] was used to identify ESBL activity. This method was performed using a combination of cefepime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), Amoxicillin (20 μg)/clavulanate (10 μg). ESBL producers were identified by reduced zone sizes to third-generation cephalosporins (ceftazidime and ceftriaxone), and expansion of these zones in the presence of an inhibitor (clavulanate). Confirmatory tests for ESBL producers were performed using oxyamino-cephalosporins/β-lactam inhibitor combinations, namely cefotaxime (30 μg)/clavulanate (10 μg) and ceftazidime (30 μg)/clavulanate (10 μg) (Additional file 1: Figure S1B). Zone sizes were measured and interpreted according to the CLSI guidelines [11].
Phenotypic AmpC activity was detected using two different tests. First, we measured zone sizes using a combination of cefoxitin and an alternative third- and fourth-generation cephalosporin, where an AmpC positive organism would be resistant to cefoxitin and exhibit reduced susceptibility to the alternative third generation cephalosporin (ceftriaxone 30 μg) and complete susceptibility to the fourth generation cephalosporin (cefepime). Secondly, we determined inducible AmpC phenotypes by assessing reduced zone sizes against a third generation cephalosporin (ceftriaxone 30 μg or ceftazidime 30 μg) in the presence of imipenem (30 μg) as an inducing substrate (Additional file 1: Figure S1A) [13, 14].
Genotypic screening of ESBL and AmpC genes
Blood isolates collected from the same patient at two different time-points with the same phenotypic AMR profile were denoted as duplicates and only one was subsequently selected for further PCR analysis. If the AMR profile of the two isolates selected differed, both were selected for PCR analysis. Multiplex PCR reactions were used to detect ESBL (bla
CTX-M subtypes) [15] and AmpC [7] genes. Other β-lactamase genes, bla
TEM [16], bla
OXA [17], and bla
SHV [16] were also detected by multiplex PCR using the following cycling conditions, initial denaturation step at 95 °C for 15 min, followed by 25 cycles of DNA denaturation at 94 °C for 30 s, primer annealing at 57 °C for 40 s, and primer extension at 72 °C for 30 s. After the last cycle, a final extension step at 72 °C for 10 min was added. PCR amplicons were examined by agarose gel (BioRad) electrophoresis made up to a concentration of 1.5% (w/v). The primers used in this study are shown in Additional file 2: Table S1.
All positive ESBL (bla
CTX-M) and AmpC (bla
CIT) PCR amplicons were sequenced to further subtype these genes. DNA was extracted using the Agencourt AMPure XP PCR purification system (Beckman Coulter) and sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied biosystems) on the 3130 genetic analyser (Applied biosystems). DNA sequences were compared against the National Centre of Biotechnology Information (NCBI) GenBank sequence database using the BLASTn algorithm and gene variants were subsequently deduced based on sequence similarity.
Statistical analysis
Statistical analysis was performed in R. Comparisons between HAI and CAI antimicrobial resistance instance rates was assessed using the Fisher’s exact test (two-tailed). A p-value < 0.05 was considered statistically significant.