A total of 327 ESBL producers were sequenced, 255 E. coli and 72 K. pneumoniae (Additional file 1). Of them, 203 E. coli (79.6%) and 55 K. pneumoniae (76.4%) were recovered from screening specimens. Sixty-nine strains were isolated from clinical specimens (E. coli 52 isolates; 25.5% and K. pneumoniae 17 isolates; 31.5%), including urine (36 isolates; 52.2%), peritoneal fluid (8 isolates; 11.6%), respiratory tract (7 isolates; 10.1%), blood stream (6 isolates; 8.7%), pus and wound (5 isolates; 7.2% each) and cerebrospinal fluid (2 isolates; 2.9%). The proportion of clinical isolates in GPED, PICU and NICU were 27.6% (55 isolates), 8.9% (8 isolates) and 15.8% (6 isolates), respectively.
Eighty-eight different sequence types (STs) were detected in E. coli, whereas 36 STs were found in K. pneumoniae. The 5 most prevalent STs in E. coli were ST131 (42 isolates; 16.5%) followed by ST38 and ST10 (21 isolates; 8.2% each), ST1193 (10 isolates; 3.9%) and ST73 (8 isolates; 3.1%). All ST131 isolates belonged to phylogroup B2. O25:H4 accounted for 23 ST131 clones (54.8%). The 3 most common STs among K. pneumoniae isolates were ST307 (7 isolates; 9.7%) followed by ST45 and ST268 (5 isolates; 6.9% each) (Additional file 1 and Additional file 2).
Eighty percent of NICU isolates, including all K. pneumoniae isolates, were detected during hospital stay. Carriage acquisition rate and infection acquisition rate in NICU during hospitalisation for the study period were 3.28/1000 patient-days and 0.82/1000 patient-days. Twenty-nine percent of PICU isolates were detected during hospitalization. Carriage acquisition rate and infection acquisition rate in PICU were 5.82/1000 patient-days and 0.76/1000 patient-days. There were no outbreaks detected in these units during the study.
Overall, non-susceptibility rates among ESBL-producing E. coli and K. pneumoniae isolates were 99.2% and 98.6% for ceftriaxone, 60.2% and 87.3% for ceftazidime, 68.8% and 73.2% for cefepime, 46.5% and 70.8% for amoxicillin/clavulanate, 12.5% and 31% for piperacillin/tazobactam, 5.1% and 9.9% for ertapenem, 3.1% and 8.5% for meropenem, 18.4% and 29.6% for gentamicin, 0.8% and 4.2% for amikacin, 41.4% and 40.8% for ciprofloxacin, 66% and 76.1% for co-trimoxazole, and 7.8% and 60.6% for nitrofurantoin. There were no significant differences in non-susceptibility rates between clinical and screening isolates by nursing unit. Of the total 69 bacterial isolates recovered from clinical specimens, 42% (29 isolates) and 20.3% (14 isolates) were non-susceptible to amoxicillin/clavulanate and piperacillin/tazobactam. By contrast, non-susceptibility among clinical isolates to ertapenem and amikacin was 3% (2 isolates) and 1.5% (1 isolate). All 29 urinary isolates of E. coli were susceptible to nitrofurantoin.
WGS revealed that all 327 isolates except one carried CTX-M genes. The predominant gene detected was blaCTX−M−15 in 287 isolates (87.8%), followed by blaCTX−M−14 and blaSHV-106 in 9 isolates each (2.8%). ESBL-producing E. coli and ESBL-producing K. pneumoniae were isolated simultaneously from 17 samples with blaCTX−M−15 detected in both species in 16 of those samples.
Sixty-seven percent of isolates co-produced one or more β-lactamases. The most common genes were blaTEM−1B and blaOXA-1 in 134 (41%) and 64 (19.6%) isolates, respectively. CTX-M-15 was present in all isolates carrying blaOXA-1 (Table 1).
We further assessed the association between co-production of TEM-1B and OXA-1 enzymes and non-susceptibility to amoxicillin/clavulanate and piperacillin/tazobactam, after excluding the confounding influence of other β-lactamases active against β-lactamase inhibitors, namely plasmid-mediated AmpC β-lactamases, (DHA-1, CMY 1/2/135), inhibitor-resistant TEM mutants (TEM 33/35) and carbapenemases (OXA-48-type and NDM-type). Non-susceptibility to amoxicillin/clavulanate was observed in 53.9% of isolates (48/89) carrying blaTEM−1B alone (P < 0.001) and 75% (15/20) carrying blaOXA−1 alone (P = 0.002), whereas non-susceptibility to piperacillin/tazobactam was observed in 5.6% of isolates (5/89) carrying blaTEM−1B alone (P = 0.5) and 25% (5/20) carrying blaOXA−1 alone (P = 0.02). Thirty-one of the 32 isolates (96.8%) carrying both enzymes simultaneously were non-susceptible to amoxicillin/clavulanate (P < 0.0001), whereas 15 (46.9%) of them were non-susceptible to piperacillin/tazobactam (P = 0.004).
At least one plasmid-mediated quinolone resistance (PMQR) gene was detected in 199 isolates (60.9%). PMQR genes found were qnr A/B/E/S in 148 isolates (45.3%), oqxAB in 68 isolates (20.8%), aac(6’)-Ib-cr in 57 isolates (17.4%) and qepA in 2 isolates (0.6%). oqxAB were mostly detected in K. pneumoniae (67/68 isolates; 98.5%). Fifteen different aminoglycoside modifying enzymes genes were detected. Ninety percent of isolates non-susceptible to gentamicin carried genes encoding AAC(3) enzymes (61/68 isolates). Conversely, only 2 (3.5%) of the 57 isolates harbouring aac(6’)-Ib-cr were non-susceptible to amikacin. All 4 isolates with high-level resistance to amikacin carried 16S rRNA methylase genes (2 rmtB and 2 armA). The simultaneous carriage of blaOXA−1, aac(6’)-Ib-cr and aac(3)-II was strongly associated (P < 0.0001). Eighty-nine percent of the isolates harbouring the dfrA17 gene also carried the aadA5 gene while all isolates harbouring dfrA12 carried aadA2 genes (Table 1).
Narrow host-range IncF plasmids were present in 278 isolates (85%). FIB was the most common F replicon found in 243 isolates (74.3%) followed by FII in 162 isolates (49.5%) and FIA 99 isolates (30.3%). In addition, 10 more incompatibility group plasmids were detected, mostly as a part of multiple combinations with F replicons. The 3 most prevalent were: IncI1 (62 isolates; 19%), IncX (27 isolates; 8.3%) and IncB/O/K/Z (25 isolates; 7.6%). On the other hand, 189 isolates (57.8%) contained col-type plasmids. RNAI (119 isolates, 36.4%), col156 (88 isolates; 26.9%) and MG828 (46 isolates; 14%) were the most common representatives within this group. Plasmid location of blaCTX−M genes could be determined in 21 isolates (Table 2). Twenty-one isolates (6.4%) did not have any plasmid detected.