Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Background: Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. Methods: V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. Results: All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Conclusions: Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

OBJECTIVES: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. METHODS: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. RESULTS: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. CONCLUSIONS: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Relationship between Penicillin-Binding Proteins Alterations and ß-Lactams Non-Susceptibility of Diseased Pig-Isolated Streptococcus suis.

Streptococcus suis is a zoonotic pathogen causing disease in both animals and humans, and the emergence of increasingly resistant bacteria to antimicrobial agents has become a significant challenge globally. The objective of this study was to investigate the genetic basis for declining susceptibility to penicillin and other ß-lactams among S. suis. Antimicrobial susceptibility testing and penicillin-binding proteins (PBP1a, PBP2a, PBP2b, and PBP2x) sequence analysis were performed on 225 S. suis isolated from diseased pigs. This study found that a growing trend of isolates displayed reduced susceptibility to ß-lactams including penicillin, ampicillin, amoxicillin/clavulanic acid, and cephalosporins. A total of 342 substitutions within the transpeptidase domain of four PBPs were identified, of which 18 substitutions were most statistically associated with reduced ß-lactams susceptibility. Almost all the S. suis isolates which exhibited penicillin-non-susceptible phenotype (71.9%) had single nucleotide polymorphisms, leading to alterations of PBP1a (P409T) and PBP2a (T584A and H588Y). The isolates may manifest a higher level of penicillin resistance by additional mutation of M341I in the 339STMK active site motif of PBP2x. The ampicillin-non-susceptible isolates shared the mutations in PBP1a (P409T) and PBP2a (T584A and H588Y) with additional alterations of PBP2b (T625R) and PBP2x (T467S). The substitutions, including PBP1a (M587S/T), PBP2a (M433T), PBP2b (I428L), and PBP2x (Q405E/K/L), appeared to play significant roles in mediating the reduction in amoxicillin/clavulanic acid susceptibility. Among the cephalosporins, specific mutations strongly associated with the decrease in cephalosporins susceptibility were observed for ceftiofur: PBP1a (S477D/G), PBP2a (E549Q and A568S), PBP2b (T625R), and PBP2x (Q453H). It is concluded that there was genetically widespread presence of PBPs substitutions associated with reduced susceptibility to ß-lactam antibiotics.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Antimicrobial Susceptibility of Streptococcus suis Isolated from Diseased Pigs in Thailand, 2018-2020.

Streptococcus suis is a porcine and zoonotic pathogen that causes severe systemic infection in humans and pigs. The treatment of S. suis infection relies on antibiotics; however, antimicrobial resistance (AMR) is an urgent global problem, pushing research attention on the surveillance of antibiotic-resistant S. suis to the fore. This study investigated the antimicrobial susceptibility of 246 S. suis strains isolated from diseased pigs in Thailand from 2018-2020. The major sources of S. suis strains were lung and brain tissues. PCR-based serotyping demonstrated that the most abundant serotype was serotype 2 or ½, followed by serotypes 29, 8, 9, and 21. To the best of our knowledge, this is the first report describing the distribution of AMR S. suis serotype 29 in diseased pigs. The antimicrobial susceptibility test was performed to determine the minimum inhibitory concentrations of 35 antimicrobial agents. The results showed that important antimicrobial agents for human use, amoxicillin/clavulanic acid, daptomycin, ertapenem, meropenem, and vancomycin, were the most effective drugs. However, a slight decrease in the number of S. suis strains susceptible to amoxicillin/clavulanic acid and vancomycin raised awareness of the AMR problem in the future. The data indicated a tendency of reduced efficacy of available veterinary medicines, including ampicillin, cefepime, cefotaxime, ceftiofur, ceftriaxone, chloramphenicol, florfenicol, gentamicin, penicillin, and tiamulin, for the treatment of S. suis infection, thus emphasizing the importance of the prudent use of antibiotics. The widespread of multidrug-resistant S. suis strains was identified in all serotypes and from different time periods and different regions of the country, confirming the emergence of the AMR problem in the diseased pig-isolated S. suis population.

Colistin Susceptibility Testing by Rapid Colistin Disk Elution Test Among Enterobacteriaceae in Low-Resource Setting.

We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-µg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 µg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-µL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

Geographic Drivers of Antimicrobial Use and Resistance in Pigs in Khon Kaen Province, Thailand.

In Thailand, pig production has increased considerably in the last decades to meet a growing demand for pork. Antimicrobials are used routinely in intensive pig production to treat infections and increase productivity. However, the use of antimicrobials also contributes to the rise of antimicrobial resistance with potential consequences for animal and human health. Here, we quantify the association between antimicrobial use and resistance rates in extensive and intensive farms with a focus on geographic proximity between farm and drugstores. Of the 164 enrolled farms, 79% reported using antimicrobials for disease prevention, treatment, or as a feed additive. Antimicrobial-resistant E. coli were present in 63% of farms. These drugs included critically important antimicrobials, such as quinolones and penicillins. Medium-scale farms with intensive animal production practices showed higher resistance rates than small-scale farms with extensive practices. Farms with drug-resistant Escherichia coli were located closer to drugstores and a had a higher proportion of disease than farms without drug-resistant E. coli. We found no association between the presence of resistance in humans and antimicrobial use in pigs. Our findings call for actions to improve herd health to reduce the need for antimicrobials and systematic training of veterinarians and drugstore owners on judicious use of antimicrobials in animals to mitigate resistance.

Exploring the Antibiotic Resistance Burden in Livestock, Livestock Handlers and Their Non-Livestock Handling Contacts: A One Health Perspective.

Antibiotics are freqeuently used in the livestock sector in low- and middle-income countries for treatment, prophylaxis, and growth promotion. However, there is limited information into the zoonotic prevalence and dissemination patterns of antimicrobial resistance (AMR) within these environments. In this study we used pig farming in Thailand as a model to explore AMR; 156 pig farms were included, comprising of small-sized (<50 sows) and medium-sized (≥100 sows) farms, where bacterial isolates were selectively cultured from animal rectal and human fecal samples. Bacterial isolates were subjected to antimicrobial susceptibility testing (AST), and whole-genome sequencing. Our results indicate extensive zoonotic sharing of antibiotic resistance genes (ARGs) by horizontal gene transfer. Resistance to multiple antibiotics was observed with higher prevalence in medium-scale farms. Zoonotic transmission of colistin resistance in small-scale farms had a dissemination gradient from pigs to handlers to non-livestock contacts. We highly recommend reducing the antimicrobial use in animals' feeds and medications, especially the last resort drug colistin.

Characterization of Klebsiella pneumoniae complex isolates from pigs and humans in farms in Thailand: population genomic structure, antibiotic resistance and virulence genes.

OBJECTIVES: To define characteristics of Klebsiella pneumoniae complex (hereafter KP) isolates from healthy pigs, farm workers and their household members in Thailand. METHODS: A total of 839 individual rectal swabs from pigs on 164 farms and 271 faecal samples of humans working on pig farms and persons living in the same household in Khon Kaen, Thailand were screened for gut colonization by KP. Genomic sequences were investigated for antibiotic resistance and virulence genes. Phylogenetic analyses were performed in addition to comparison with isolates from previous studies from Thailand. RESULTS: KP was detected in approximately 50% of pig and human samples. In total, 253 KP isolates were obtained: 39% from pigs, 34% from farmers and 26% from individuals living on the same farm but without animal contact. MLST revealed high genetic diversity with 196 different STs distributed over four phylogroups (Kp1 to Kp4). Low prevalence of ESBL-KP (7.5%) and colistin-resistant KP (3.2%) was observed among pigs and humans. Remarkably, four convergent MDR and hypervirulent strains were observed: one from pigs (ST290) and three from humans [ST35, ST3415 (strain 90CP1), ST17 (strain 90CM2)]. Sharing of KP clones among pigs and humans was identified for some STs including ST4788, ST661, ST3541 and ST29. CONCLUSIONS: The study indicated a low prevalence of ESBL and mcr genes among KP isolated from pigs and healthy humans in Thailand and suggested the possibility of zoonotic transmission for a subset of circulating KP clones.

Antibiotic use in pig farms at different levels of intensification-Farmers' practices in northeastern Thailand.

Understanding the patterns and drivers of antibiotic use in livestock is crucial for tailoring efficient incentives for responsible use of antibiotics. Here we compared routines for antibiotic use between pig farms of two different levels of intensification in Khon Kaen province in Thailand. Among the 113 family-owned small-scale farms (up to 50 sows) interviewed did 76% get advice from the pharmacy about how to use the antibiotics and 84% used it primarily for treating disease. Among the 51 medium-scale-farms (100-500 sows) belonging to two companies did 100% get advice about antibiotic use from the company's veterinarian (P<0.0001) and 94% used antibiotics mostly as disease preventive measure (P<0.0001). In 2 small scale farms 3rd generation cephalosporins, tylosin or colistin were used; antibiotics belonging to the group of highest priority critically important antimicrobials for human medicine. Enrofloxacin, belonging to the same group of antimicrobials, was used in 33% of the small-scale and 41% of the medium-scale farms. In the latter farms, the companies supplied 3-4 antibiotics belonging to different classes and those were the only antibiotics used in the farms. The median and mean estimated expenditure on antibiotics per sow was 4.8 USD (IQR = 5.8) for small-scale farms and 7 USD and 3.4 USD for the medium-scale farms belonging to the two respective companies. Our observations suggest to target the following areas when pig farming transitions from small-scale to medium-scale: (i) strengthening access to professional animal health services for all farmers, (ii) review of the competence and role of veterinary pharmacies in selling antibiotics and (iii) adjustment of farming company animal health protocols towards more medically rational use of antibiotics.

PmrB mutations including a novel 10-amino acid repeat sequence insertion associated with low-level colistin resistance in carbapenem-resistant Acinetobacter baumannii.

The global emergence of colistin resistance in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates is a serious public health concern. We therefore aimed to investigate colistin resistance mechanisms in 5 colistin-resistant (COL-R) CRAB isolates collected from Thai patients in 2016 by whole genome sequencing (WGS) compared with those of 5 colistin-intermediate (COL-I) CRAB isolates from the same period. All isolates were subjected to antimicrobial susceptibility testing, efflux pump inhibitor-based test and WGS. Mutations in known genes associated with colistin resistance were analyzed and deleterious mutations were then predicted by PROVEAN tool. The 10 CRAB isolates carried blaOXA-23 with the addition of blaOXA-58 in 1 isolate. All COL-R isolates exhibited colistin MICs of 4 µg/mL except for 1 isolate with that of 16 µg/mL. They belonged to ST2, ST16, ST23, ST164 and ST215, whereas the COL-I isolates with colistin MICs of ≤0.25-1 µg/mL were ST2, ST164 and ST215. Neither increased efflux pump activity nor mcr gene was found in any COL-R isolate. Three COL-R isolates contained different PmrB variants: a novel 10-amino acid (aa) repeat sequence insertion, VILGCILIFS between positions 27 and 28 (S27_A28insVILGCILIFS) in transmembrane domain (TM); a 1-aa insertion, alanine between positions 162 and 163 (A162_I163insA) in TM; and a 1-aa substitution, A226T in histidine kinase domain. One COL-R isolate possessed PmrA variant with A80V substitution. These alterations were predicted as deleterious. Mechanisms of colistin resistance in the remaining COL-R isolate were still unknown. In conclusion, the alterations in both PmrB and PmrA were predicted and suggested as initial mutations responsible for low-level colistin resistance in our CRAB isolates. Under selective pressure, these isolates may exhibit higher level colistin resistance by the additional mutations, leading to more therapeutic difficulties.

Antimicrobial Resistance in Fecal Escherichia coli from Humans and Pigs at Farms at Different Levels of Intensification.

The overall aim of the current study was to test the hypotheses that (i) antibiotic resistance in bacteria were more frequent in clinically health pigs in intensified company owned, medium-scale farms (MSFs) (100-500 sows) than in pigs in family-owned, small-scale farms (SSFs) (1-50 sows) and (ii) that farmers working at the MSFs were more prone to attain antibiotic resistant bacteria than farmers working at SSFs. The study was conducted in North-Eastern Thailand, comprising fecal Escherichia coli isolates from pigs, farmers working with the pigs (contact humans) and persons living in the same household as the farmer (non-contact humans) at 51 MSFs and 113 SSFs. Samples from all farms were also screened for methicillin-resistant staphylococcus aureus (MRSA), which was not detected in pig samples, but was found in one human sample. Susceptibility was tested by disc-diffusion for seven antibiotics commonly used in the study area. Resistance in pig isolates from MSFs were more frequent for chloramphenicol which (P < 0.001), trimethoprim/sulfamethoxazole (P < 0.001) and gentamicin (P < 0.05) compared with isolates from SSFs, whereas the opposite was true for tetracycline (P < 0.01). Resistance in the human isolates was lower than those in the isolates from pigs for tetracycline, trimethoprim/sulfamethoxazole and chloramphenicol (P < 0.001). The frequency of resistance in the contact human samples from SSFs and MSFs did not differ. There was no difference between isolates from contact and non-contact humans for any of the tested antibiotics. Multidrug resistance in isolates from pigs was 26%, significantly higher (P < 0.01) than the 13% from humans. The data indicate that (i) resistance to antibiotics, including those critical and highly important for human medicine, were more common in fecal E. coli from pigs at the MSFs than at the SSFs, whereas (ii) the resistance in fecal E. coli from pig farmers seemed not to be influenced by the level of intensification of the farm they were working at.

Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital.

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC50 and MIC90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 µg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC50 and MIC90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried blaOXA-23-like. The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

Molecular Characterization of Carbapenemase-Nonproducing Clinical Isolates of Escherichia coli (from a Thai University Hospital) with Reduced Carbapenem Susceptibility.

Twelve nonreplicate carbapenemase-negative ertapenem (ETP)-nonsusceptible (CNENS) Escherichia coli isolates obtained at a Thai university hospital between 2010 and 2014 were characterized and compared with 2 carbapenemase-producing E. coli isolates from the same hospital. Eight unique pulsed-field gel electrophoresis patterns were obtained. All the isolates produced CTX-M-15 ß-lactamase and 2 either coexpressed CMY-2 cephalosporinase or showed increased efflux pump activity. Amino acid sequence analysis revealed that an OmpF defect (in 7 isolates) due to mutations generating truncated proteins or an IS1 insertion was more prevalent than a defect in OmpC was (no truncated proteins detected). Seven out of 10 isolates possessing OmpC variants with any OmpF defect were weakly ETP-resistant (minimum inhibitory concentrations [MICs] of 1-4 µg/mL) and imipenem (IPM)- and meropenem (MEM)-susceptible (MICs 0.125-0.5 µg/mL). Two isolates with ompC PCR-negative results and an OmpF defect showed higher carbapenem MICs (8-32, 1-8, and 1-4 µg/mL for ETP, IPM, and MEM, respectively) with the highest MICs associated with the additional efflux pump activity. Both carbapenemase producers possessing CTX-M-15 and a porin background identical to that in the CNENS isolates showed ETP, IPM, and MEM MICs of 128-256, 8, and 2-32 µg/mL, respectively. These findings suggest that a porin defect combined with CTX-M-15 production is the major mechanism of low carbapenem susceptibility among our CNENS isolates, which have potential to become strongly carbapenem-resistant because of additional carbapenemase or efflux pump activities.

High-level carbapenem-resistant OXA-48-producing Klebsiella pneumoniae with a novel OmpK36 variant and low-level, carbapenem-resistant, non-porin-deficient, OXA-181-producing Escherichia coli from Thailand.

Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future.