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Background Romania is one of the European countries reporting very high antimicrobial resistance (AMR) rates and consumption of antimicrobials. We aimed to characterize the AMR profiles and clonality of 304 multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) strains isolated during two consecutive years (2018 and 2019) from hospital settings, hospital collecting sewage tanks and the receiving wastewater treatment plants (WWTPs) located in the main geographical regions of Romania. Methods The strains were isolated on chromogenic media and identified by MALDI-TOF-MS. Antibiotic susceptibility testing and confirmation of ESBL- and CP- producing phenotypes and genotypes were performed. The genetic characterization also included horizontal gene transfer experiments, whole-genome sequencing (WGS), assembling, annotation and characterization. Results Both clinical and aquatic isolates exhibited high MDR rates, especially the Ab strains isolated from nosocomial infections and hospital effluents. The phenotypic resistance profiles and MDR rates have largely varied by sampling point and geographic location. The highest MDR rates in the aquatic isolates were recorded in Galați WWTP, followed by Bucharest. The Ab strains harbored mostly blaOXA-23, blaOXA-24, blaSHV, blaTEM and blaGES, while Pa strains blaIMP, blaVIM, blaNDM, blaVEB, blaGES and blaTEM, with high variations depending on the geographical zone and the sampling point. The WGS analysis revealed the presence of antibiotic resistance genes (ARGs) to other antibiotic classes, such as aminoglycosides, tetracyclines, sulphonamides, fosfomycin, phenicols, trimethoprim-sulfamethoxazole as well as class 1 integrons. The molecular analyses highlighted: (i) The presence of epidemic clones such as ST2 for Ab and ST233 and ST357 for Pa; (ii) The relatedness between clinical and hospital wastewater strains and (iii) The possible dissemination of clinical Ab belonging to ST2 (also proved in the conjugation assays for blaOXA-23 or blaOXA-72 genes), ST79 and ST492 and of Pa strains belonging to ST357, ST640 and ST621 in the wastewaters. Conclusion Our study reveals the presence of CP-producing Ab and Pa in all sampling points and the clonal dissemination of clinical Ab ST2 strains in the wastewaters. The prevalent clones were correlated with the presence of class 1 integrons, suggesting that these isolates could be a significant reservoir of ARGs, being able to persist in the environment.

Introduction Infections due to extended spectrum beta-lactamase producing organisms (ESBL) have emerged as the leading cause of sepsis among hospitalized neonates in Botswana and much of sub-Saharan Africa and south Asia. Yet, ESBL reservoirs and transmission dynamics within the neonatal intensive care unit (NICU) environment are not well-understood. This study aimed to assess the efficiency and feasibility of a chromogenic-culture-media-based environmental sampling approach to characterize the ESBL bioburden within a NICU. Methods A series of four point-prevalence surveys were conducted at a 36-bed NICU at a public tertiary referral hospital in Botswana from January-June 2021. Samples were collected on 4 occasions under semi-sterile technique using 1) flocked swabs & templates (flat surfaces); 2) sterile syringe & tubing (water aspiration); and 3) structured swabbing techniques (hands & equipment). Swabs were transported in physiological saline-containing tubes, vortexed, and 10 µL was inoculated onto chromogenic-agar that was selective and differential for ESBL (CHROMagar™ ESBL, Paris, France), and streaking plates to isolate individual colonies. Bacterial colonies were quantified and phenotypically characterized using biochemical identification tests. Results In total, 567 samples were collected, 248 (44%) of which grew ESBL. Dense and consistent ESBL contamination was detected in and around sinks and certain high-touch surfaces, while transient contamination was demonstrated on medical equipment, caregivers/healthcare worker hands, insects, and feeding stations (including formula powder). Results were available within 24–72 h of collection. To collect, plate, and analyse 50 samples, we estimated a total expenditure of $269.40 USD for materials and 13.5 cumulative work hours among all personnel. Conclusions Using basic environmental sampling and laboratory techniques aided by chromogenic culture media, we identified ESBL reservoirs (sinks) and plausible transmission vehicles (medical equipment, infant formula, hands of caregivers/healthcare workers, & insects) in this NICU environment. This strategy was a simple and cost-efficient method to assess ESBL bioburden and may be feasible for use in other settings to support ongoing infection control assessments and outbreak investigations.

Background Carbapenem-resistant Enterobacterales (CRE), currently listed by the World Health Organization (WHO) as top priority critical pathogens, are a major global menace to human health. In low- and middle-income countries (LMICs) the threat is mounting fueled by selective pressures caused by antibiotic abuse and inadequate diagnostic resources. Methods This study phenotypically and genotypically characterized carbapenem resistance among 115 Enterobacterales isolates including 76 Klebsiella (K.) pneumoniae, 19 Escherichia (E.) coli, 14 Shigella (S.) sonnei, 5 Enterobacter (E.) cloacae, and 1 Proteus (P.) mirabilis. Results Ninety-three isolates (80.9%) were carbapenem-resistant with an alarming 57.5% carbapenem non-susceptibility in isolates collected from the outpatient department. Molecular characterization of the carbapenemases (CPases) encoding genes showed that blaNDM (80.5%) was the most prevalent; it was detected in 62 isolates (54 K. pneumoniae, 6 E. coli and 2 S. sonnei), followed by blaVIM (36.4%) which was observed in 28 isolates (24 K. pneumoniae, 3 E. coli and 1 E. cloacae). Other CPases included blaKPC (28.6%; in 20 K. pneumoniae, 1 E. coli and 1 S. sonnei), blaOXA-48 (26%; in 17 K. pneumoniae, 1 E. coli,1 E. cloacae and 1 P. mirabilis), blaIMP (6.5%; in 5 K. pneumoniae) and blaSPM (1.3%; in K. pneumoniae). Notably more than half of the Enterobacterales isolates (54.5%) co-harboured more than one CPase-encoding gene. Co-existence of blaNDM and blaVIM genes was the most dominant (31.2%), followed by association of blaNDM and blaKPC (24.7%), then blaVIM and blaKPC (13%). Moreover, the effects of different genotypes on meropenem MIC values were assessed, and a statistically significant difference between the genotype (Ambler classes A and B) and the genotype (Ambler classes B and D) was recorded. Conclusion The current findings may serve for a better understanding of the context of CRE in Egypt, associated drivers and CPases.

Background Approximately 11% of patients colonized with extended-spectrum beta-lactamase producing Enterobacterales (ESBL-PE) are colonized with more than one ESBL-producing species. We investigated risk factors associated with colonization with multiple ESBL-PE species. Methods We performed a case-case–control study at the University Hospital Basel, Switzerland, including hospitalized patients colonized with ESBL-PE between 01/2008 and 12/2018. Patients colonized with multiple species of ESBL-PE during the same hospitalization were assigned to group 1. Group 2 consisted of patients with ESBL-PE and a newly acquired ESBL-PE-species identified during subsequent hospitalization. Controls (i.e., group 3) were patients with only one species of ESBL-PE identified over multiple hospitalizations. Controls were frequency-matched 3:1 to group 2 cases according to time-at-risk (i.e., days between ESBL-PE detection during first and subsequent hospitalizations) to standardize the duration of colonization. ESBL was identified with phenotypic assay and the presence of ESBL genes was confirmed by whole genome sequencing. Results Among 1559 inpatients, 154 cases met eligibility criteria (67 in group 1, 22 in group 2, 65 in group 3). International travel within the previous 12 months (OR 12.57, 95% CI 3.48–45.45, p Conclusion These findings point to strain-related factors being the main drivers of co-colonization with different ESBL-PE and may support stratification of infection prevention and control measures according to ESBL-PE species/strains.

BackgroundEscherichia colisequence type (ST) 131 H30 is an emerging multidrug resistant subclone, known to spread and cause outbreaks in long-term care facilities (LTCFs).Objectives and methodsFrom 2010 through 2020, we performed 11 yearly surveillance studies for determining the prevalence of digestive carriage of ESBL-producingE. coli(ESBL-EC) among residents in a university-affiliated LCTF. Sequencing and genotyping of selected isolates were performed to characterize temporal trends in the prevalence and epidemic potential of ESBL-EC subclones, and for evaluating a potential rebound effect following discontinuation of contact precautions for ESBL-EC carriers in January 2019.ResultsThis study included 2′403 LTCF residents, with 252 (10.5%) positive for ESBL-EC. Among the 236 ESBL-EC isolates available for typing, 58.0% belonged to the ST131 lineage, including 94/137 (68.6%) ST131 H30 isolates. An increasing yearly prevalence was observed for ESBL-EC (from 4.6 to 9.4%;p = 0.11), but not for the ST131 H30 subclone, which peaked in 2015 and declined thereafter. Multiple previously unnoticed ESBL-EC outbreaks occurred in the LTCF. Since 2018, we noted the clonal expansion of a rare ST131 H89 subclone (O16:H5) harboring CTX-M-14 and CTX-M-24. No rebound effect was observed in ESBL-EC prevalence nor in the different subclones following discontinuation of contact precautions for ESBL-EC carriers since 2019.ConclusionClonal fluctuation was observed for ST131 H30 ESBL-EC with a current decline in prevalence. Surveillance should include the evolution of ST131 non-H30 subclones, which may spread in LTCFs. Our findings suggest that discontinuation of contact precautions for ESBL-EC carriers in LTCFs may be safely implemented, in support of European recommendations to limit ESBL-producing Enterobacteriaceae control measures in endemic settings to non-E. coli.

Background Infections caused by Enterobacteriaceae are mainly treated with the β-lactam antibiotics, nevertheless, the emergence of species with plasmid-borne β-lactamases has decreased the efficacy of these antibiotics. Therefore, continuing studies on the resistance pattern of different regions is important for assessment of proper antimicrobial therapy protocols. The study aimed to characterize extended-spectrum β-lactamase (ESBL) and AmpC β –lactamase (AmpC) producing Enterobacteriaceae isolated from community-acquired UTIs in Egypt. Methods Out of 705 urine samples, 440 Enterobacteriaceae isolates were investigated to detect ESBL and AmpC β -lactamases producers by phenotypic and molecular methods. Results Out of 440 Enterobacteriaceae isolates, 311 were identified as ESBL producers by phenotypic testing. ESBL genes were detected in 308 isolates. BlaCTX-M-type was the most prevalent 254 (81.6%), out of them blaCTXM-15 was the commonest (152, 48.8%) followed by blaCTX-M-1 (140, 45%), blaCTX-M-8 (72, 23.1%) and lastly blaCTX-M-2 (4, 1.3%). blaTEM gene also was detected in a high rate (189, 60.7%). Two hundred and thirty-five (75.5%) of ESBL producers harbored blaCTX-M in combination with blaTEM and/or blaSHV genes. Multiple drug resistance in the ESBL-producers was significantly (P -lactamase genes, DHA gene was the predominant gene (15, 42.3%). CIT and MOX genes were also present, but in a low rate (5, 14.2% and 4, 11.4%) respectively. Co-existence of multiple AmpC genes was detected exclusively in K. pneumoniae isolates. E. coli isolates harbored DHA gene only. However, FOX gene was not detected in the study isolates. Seventeen of isolates carrying AmpC genes were also positive for ESBL genes. Conclusion The study shows that the prevalence of ESBL producing Enterobacteriaceae spread in south Egypt is alarming, however AmpC β -lactamase production is not so high.

Background The aim of this study was to detect CMY-type beta-lactamases in E. coli isolates obtained from paediatric patients. Methods In total, 404 infection-causing E. coli isolates resistant to third and fourth generation cephalosporins (3GC, 4GC) were collected from paediatric patients over a 2 years period. The identification and susceptibility profiles were determined with an automated microbiology system. Typing of blaCMY and other beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaVIM, blaIMP, blaKPC, blaNDM, blaOXA and blaGES) was realized by PCR and sequencing. Phenotypic detection of AmpC-type enzymes was performed using boronic acid (20 mg/mL) and cloxacillin (20 mg/mL) as inhibitors, and the production of extended-spectrum beta-lactamases was determined with the double-disk diffusion test with cefotaxime (CTX) and ceftazidime (CAZ) discs alone and in combination with clavulanic acid. The CarbaNP test and modified carbapenem inhibition method (mCIM) were used for isolates with decreased susceptibility to carbapenems. The clonal origin of the isolates was established by pulsed-field gel electrophoresis (PFGE), phylotyping method and multilocus sequence typing. Results CMY-type beta-lactamases were detected in 18 isolates (4.5%). The allelic variants found were CMY-2 (n = 14) and CMY-42 (n = 4). Of the E. coli strains with CMY, the AmpC phenotypic production test was positive in 11 isolates with cloxacillin and in 15 with boronic acid. ESBL production was detected in 13 isolates. Coexistence with other beta-lactamases was observed such as CTX-M-15 ESBL and original spectrum beta-lactamases TEM-1 and TEM-190. In one isolate, the CarbaNP test was negative, the mCIM was positive, and OXA-48 carbapenemase was detected. Phylogroup A was the most frequent (n = 9) followed by B2, E and F (n = 2, respectively), and through PFGE, no clonal relationship was observed. Eleven different sequence types (ST) were found, with ST10 high-risk clone being the most frequent (n = 4). Seventy-two percent of the isolates were from health care-associated infections; the mortality rate was 11.1%. Conclusions This is the first report in Mexico of E. coli producing CMY isolated from paediatric patients, demonstrating a frequency of 4.5%. In addition, this is the first finding of E. coli ST10 with CMY-2 and OXA-48.

Background Epidemiological studies suggested that determinants for antibiotic resistance have originated in aquaculture. Recently, the integrated agriculture-aquaculture system has been implemented, where fish are raised in ponds that receive agriculture drainage water. The present study aims to investigate the occurrence of β-lactamase and carbapenemase-producing Enterobacteriaceae in the integrated agriculture-aquaculture and the consequent public health implication. Methods Samples were collected from fish, fishpond water inlets, tap water, outlet water, and workers at sites of integrated agriculture-aquacultures. Samples were also taken from inhabitants of the aquaculture surrounding areas. All samples were cultured on MacConkey agar, the Enterobacteriaceae isolates were tested for susceptibility to cephalosporins and carbapenems, and screened for blaCTX-M-15, blaSHV, blaOXA-1, blaTEM, blaPER-1, blaKPC, blaOXA-48, and blaNDM. Strains having similar resistance phenotype and genotype were examined for the presence of Incompatible (Inc) plasmids. Results A major proportion of the Enterobacteriaceae isolates were resistant to cephalosporins and carbapenems. Among the 66 isolates from fish, 34 were resistant to both cephalosporin and carbapenem groups, 26 to carbapenems alone, and 4 to cephalosporins alone. Of the 15 isolates from fishpond water inlets, 8 showed resistance to both groups, 1 to carbapenems alone, and 5 to cephalosporins alone. Out of the 33 isolates from tap water, 17 were resistant to both groups, and 16 to cephalosporins alone. Similarly, of the 16 outlet water isolates, 10 were resistant to both groups, and 6 to cephalosporins alone. Furthermore, of the 30 examined workers, 15 carried Enterobacteriaceae resistant strains, 10 to both groups, and 5 to cephalosporins alone. Similar strains were isolated from the inhabitants of the aquaculture surrounding areas. Irrespective of source of samples, strains resistant to all examined antibiotics, carried predominantly the carbapenemase gene blaKPC either alone or with the β-lactamase genes (blaCTX-M-15, blaSHV, blaTEM, and blaPER-1). The isolates from fish, water, and workers harboured a wide-range of multi-drug-resistance Inc. plasmids, which were similar among all isolates. Conclusion The present findings suggest transmission of the resistance genes among Enterobacteriaceae strains from different sources. This reiterates the need for control strategies that focus on humans, animals, water, and sewage systems to solve the antibiotic resistance problem.

Background Treatment of pandrug-resistant isolates often necessitates combination therapy. Checkerboard synergy and time-killing assay tests were performed to evaluate the benefits of a triple combination with meropenem, ertapenem, and colistin against 10 colistin-resistant K. pneumoniae clinical isolates harboring different β-lactamases. (blaOXA-48, blaNDM). Materials and methods In this study, ertapenem and meropenem (ERT/MEM), meropenem and colistin (MEM/COL), ertapenem, meropenem and colistin (ERT/MEM/COL) combinations were tested using checkerboard techniques and time-kill assays of each antibiotic alone and in combination against 10 colistin-resistant clinical K. pneumoniae isolates. An analysis of K. pneumoniae isolate B6 using a scanning electron microscope revealed morphologic changes in the cell surface after treatment with each antibiotic both alone and in combination. The whole genome of K. pneumoniae KPNB1 was sequenced using an Ion Torrent PGM sequencer. Results According to the checkboard results, synergistic combinations were observed with ertapenem/meropenem (5/10 isolates), meropenem/colistin (7/10) and ertapenem/meropenem/colistin (9/10); no antagonism was observed for all combinations. For the time-kill assay results; synergism and bactericidal effects were observed with meropenem/colistin (10/10) and with ertapenem/meropenem/colistin (10/10) combinations, and an indifference effect was observed with the ertapenem and meropenem (10/10) combination. Strain number 1 was found 100% identical to Klebsiella pneumoniae subsp. pneumoniae HS11286 according to the outcomes of complete genome sequence analysis, and the strain carried the genes blaOXA-181, blaCTXM-15, blaNDM, arr-3, aac (6′)-Ib-cr, rmtF, and catB1. Conclusion Using double carbapenem antibiotics with colistin could be a potential alternative to treat colistin and carbapenem-resistant K. pneumoniae. The present study is the first Turkish report of OXA–181-type carbapenemase causing colistin resistance.

Background The World Health Organization (WHO) recently classified Enterobacteriaceae resistance to third-generation cephalosporin into the group of pathogens with critical criteria for future research. Methods A study to assess the antibiogram and beta-lactamase genes among the cefotaxime resistant E. coli (CREc) from a South African wastewater treatment plant (WWTP) was conducted using standard phenotypic and molecular biology characterization methods. Results Approximate total E. coli (TEc) concentration (log10 CFU/mL) ranged between 5.7 and 6.8 among which cefotaxime resistant E. coli were between 1.8 and 4.8 (log10 CFU/mL) for cefotaxime antibiotic concentration of 4 and 8 mg/L in the influent samples. Effluent samples, heavily influenced by the chlorination had only 0.3 log10 CFU/mL of TEc. Fifty-one cefotaxime resistant isolates were selected out of an overall of 75 isolates, and subjected to a new round of testing, with a follow up of 36 and 48 isolates for both colistin and gentamicin, respectively as guided by initial results. Selected CREc exhibited resistance to amoxicillin-clavulanic acid (35.3%; n = 51), colistin sulphate (76.5%; n = 36), ciprofloxacin (47.1%; n = 51), gentamicin (87.5%; n = 48) and intermediate-resistance to meropenem (11.8%; n = 51). Extended spectrum-beta-lactamase genes detected, viz.: blaCTX-M (52.6%; n = 38) and blaTEM (84.2%; n = 38) and concurrent blaCTX-M + blaTEM (36.8%; n = 38), but no blaSHV was detected. Carbapenem resistance genes, blaKPC-2 (15.8%; n = 38), blaOXA-1 (57.9%; n = 38), blaNDM-1 (15.8%; n = 38) were also detected. Approximately, 10.5 - 36.8% (n = 38) co-occurrence of two or more beta-lactamase genes was detected in some isolates. Out of the selected number (n = 30), 7(23.3%) were enterotoxigenic E. coli (ETEC), 14 (46.7%) were Enteroaggregative E. coli (EAEC), but no enteropathogenic E. coli (EPEC) was detected. Conclusion Resistance to cefotaxime and the presence of a wide range of beta-lactamase genes exposed the potential risks associated with these pathogens via occupational and domestic exposure during the reuse of treated wastewater.

Background The spread of carbapenemase-producing Enterobacteriaceae (CPE) in the Southwest Indian Ocean area (SIOA) is poorly documented. Reunion Island is a French overseas territory located close to Madagascar and connected with Southern Africa, Indian sub-continent and Europe, with several weekly flights. Here we report the results of the CPE surveillance program in Reunion Island over a six-year period. Methods All CPE were collected between January 2011 and December 2016. Demographics and clinical data of the carrier patients were collected. We determined their susceptibility to antimicrobials, identified the carbapenemases and ESBL by PCR and sequencing, and explored their genetic relationship using pulsed-field gel electrophoresis and multi-locus sequence typing. Results A total of 61 CPEs isolated from 53 patients were retrieved in 6 public or private laboratories of the island. We found that 69.8% of CPE patients were linked to a foreign country of SIOA and that almost half of CPE cases (47.2%) reached the island through a medical evacuation. The annual number of CPE cases strongly increased over the studied period (one case in 2011 vs. 21 cases in 2016). A proportion of 17.5% of CPE isolates were non-susceptible to colistin. blaNDM was the most frequent carbapenemase (79.4%), followed by blaIMI (11.1%), and blaIMP-10 (4.8%). Autochtonous CPE cases (30.2%) harboured CPE isolates belonging to a polyclonal population. Conclusions Because the hospital of Reunion Island is the only reference healthcare setting of the SIOA, we can reasonably estimate that its CPE epidemiology reflects that of this area. Mauritius was the main provider of foreign CPE cases (35.5%). We also showed that autochthonous isolates of CPEs are mostly polyclonal, thus unrelated to cross-transmission. This demonstrates the local spread of carbapenemase-encoding genes (i.e. blaNDM) in a polyclonal bacterial population and raises fears that Reunion Island could contribute to the influx of NDM-carbapenemase producers into the French mainland territory.

Background Pseudomonas aeruginosa is a common pathogen causing hospital-acquired infections. Carbapenem resistance in P. aeruginosa is either mediated via a combination of efflux pumps, AmpC overexpression, and porin loss, or through an acquired carbapenemase. Carbapenemase-producing P. aeruginosa (CPPA) strains are known to cause outbreaks and harbour a reservoir of mobile antibiotic resistance genes, however, few molecular surveillance data is available. The aim of this study was to analyse the prevalence and epidemiology of CPPA in three German medical centres from 2015 to 2017. Methods Identification and susceptibility testing were performed with VITEK 2 system. P. aeruginosa non-susceptible to piperacillin, ceftazidime, cefepime, imipenem, meropenem and ciprofloxacin (4MRGN according to the German classification guideline) isolated from 2015 to 2017 were analysed. A two-step algorithm to detect carbapenemases was performed: phenotypic tests (EDTA- and cloxacillin-combined disk tests) followed by PCR, Sanger sequencing, and eventually whole genome sequencing. CPPA isolates were further genotyped by RAPD and PFGE. In-hospital transmission was investigated using conventional epidemiology. Results Sixty two P. aeruginosa isolates were available for further analysis, of which 21 were CPPA as follows: blaVIM-1 (n = 2), blaVIM-2 (n = 17), blaNDM-1/blaGES-5 (n = 1) and the newly described blaIMP-82 (n = 1). CPPA were mostly hospital-acquired (71.4%) and isolated on intensive care units (66.7%). All (except one) were from the tertiary care centre. PFGE typing revealed one large cluster of VIM-2-producing CPPA containing 13 isolates. However, using conventional epidemiology, we were only able to confirm three patient-to-patient transmissions, and one room-to-patient transmission, on several intensive care units. Conclusions These data give insight into the epidemiology of CPPA in three centres in Germany over a period of 3 years. Carbapenemases are a relevant resistance mechanism in 4MRGN-P. aeruginosa, illustrated by genetically related VIM-2-producing strains that seem to be endemic in this region. Our data suggest that infection control measures should especially focus on controlling transmission on the ICU and support the need for a local molecular surveillance system.

BackgroundNosocomial infections caused by multi-drug resistantEnterobacteriaceaeare a global public health threat that ought to be promptly identified, reported, and addressed accurately. Many carbapenem-resistantEnterobacteriaceae-associated genes have been identified in Saudi Arabia but not the endemicKlebsiella pneumoniaecarbapenemases (KPCs), which are encoded byblaKPC-typegenes. KPCs are known for their exceptional spreading potential.MethodsWe collectedn = 286 multi-drug resistant (MDR)Klebsiella spp.isolates as part of screening for resistant patterns from a tertiary hospital in Saudi Arabia between 2014 and 2018. Antimicrobial susceptibility testing was carried out using both VITEK II and the broth microdilution of all collected isolates. Detection of resistance-conferring genes was carried out using Illumina whole-genome shotgun sequencing and PacBio SMRT sequencing protocols.ResultsA Carbapenem-resistantEnterobacteriaceae(CRE)Klebsiella quasipneumoniaesubsp.similipneumoniaestrain was identified as a novel ST-3510 carrying ablaKPC-2carbapenemase encoding gene. The isolate, designated as NGKPC-421, was obtained from shotgun Whole Genome Sequencing (WGS) surveillance of 286 MDRKlebsiella spp. clinical isolates. The NGKPC-421 isolate was collected from a septic patient in late 2017 and was initially misidentified asK. pneumoniae. The sequencing and assembly of the NGKPC-421 genome resulted in the identification of a putative ~ 39.4 kb IncX6 plasmid harboring ablaKPC-2gene, flanked by transposable elements (ISKpn6-blaKPC-2–ISKpn27).ConclusionThis is the first identification of a KPC-2-producing CRE in the Gulf region. The impact on this finding is of major concern to the public health in Saudi Arabia, considering that it is the religious epicenter with a continuous mass influx of pilgrims from across the world. Our study strongly highlights the importance of implementing rapid sequencing-based technologies in clinical microbiology for precise taxonomic classification and monitoring of antimicrobial resistance patterns.

Background We report a recurrent outbreak of postoperative infections with extended-spectrum β-lactamase (ESBL)–producing E. cloacae complex in cardiac surgery patients, describe the outbreak investigation and highlight the infection control measures. Methods Cases were defined as cardiac surgery patients in Ghent University Hospital who were not known preoperatively to carry ESBL-producing E. cloacae complex and who postoperatively had a positive culture for this multiresistant organism between May 2017 and January 2018. An epidemiological investigation, including a case-control study, and environmental investigation were conducted to identify the source of the outbreak. Clonal relatedness of ESBL-producing E. cloacae complex isolates collected from case patients was assessed using whole-genome sequencing–based studies. Results Three separate outbreak episodes occurred over the course of 9 months. A total of 8, 4 and 6 patients met the case definition, respectively. All but one patients developed a clinical infection with ESBL-producing E. cloacae complex, most typically postoperative pneumonia. Overall mortality was 22% (4/18). Environmental cultures were negative, but epidemiological investigation pointed to transesophageal echocardiography (TEE) as the outbreak source. Of note, four TEE probes showed a similar pattern of damage, which very likely impeded adequate disinfection. The first and second outbreak episode were caused by the same clone, whereas a different strain was responsible for the third episode. Conclusions Health professionals caring for cardiac surgery patients and infection control specialists should be aware of TEE as possible infection source. Caution must be exercised to prevent and detect damage of TEE probes.

Background Extended-spectrum beta-lactamases-producing Enterobacterales (ESBL-E) are disseminating worldwide especially in Intensive Care Units (ICUs) and are responsible for increased health costs and mortality. The aims of this work were to study ESBL-E dissemination in ICU and to assess the impact of ESBL-E fecal carriage on subsequent infections during a non-outbreak situation. Methods We therefore screened every patient at admission then once a week in a medical ICU between January and June 2015. Each ESBL-E isolate was characterized by ESBL genes PCR amplification and the clonal dissemination was assessed by Pulsed-Field Gel Electrophoresis (PFGE). Results Among the 608 screened patients, 55 (9%) were colonized by ESBL-E. Forty-four isolates were available for further analysis. Most of them (43/44, 98%) contained a ESBL gene from the CTX-M group. Only one case of ESBL-E cross-transmission occurred, even for acquired ESBL-E colonization. Subsequent infection by ESBL-E occurred in 6/55 (11%) patients and infecting ESBL-E strains were the colonizing ones. ESBL-E faecal carriage had a negative predictive value of 100% and a positive predictive value of 40% to predict ESBL-E ventilator associated-pneumonia (VAP). Alternatives to carbapenems consisting in piperacillin-tazobactam, ceftolozane-tazobactam and ceftazidime-avibactam were all active on this panel of ESBL-E. Conclusions ESBL-E expansion and acquisition in ICU in a non-outbreak situation are not any more fully explained by cross-transmission. Mechanisms underlying ESBL-E dissemination in ICU are still to investigate. Interestingly, as far as we know, our study demonstrates for the first time by PFGE that the colonizing strain is indeed the infecting one in case of subsequent ESBL-E infection. Nevertheless, subsequent ESBL-E infection remains a rare event conferring poor positive predictive value for ESBL-E colonization to predict ESBL-E VAP. Relevance of systematic ESBL-E faecal screening at ICU admission and during ICU stay needs further investigation.

Background Qnr genes are known to confer a low-level resistance to fluoroquinolone in Enterobacteriaceae. They are often found on the same resistance plasmids as extended spectrum β-lactamase (ESBL) and constitute the most common antibiotic resistance mechanism. This study aimed to detect the presence of qnr genes in ESBL-producing E. coli and Klebsiella spp. Methods From May 2013 to July 2015, 91 E. coli and 64 Klebsiella spp. strains with phenotypic resistance to quinolone were collected from several specimens and analyzed for the detection of qnrA, qnrB, qnrS genes and the β-lactamase resistance genes (blaCTX-M, blaTEM, blaSHV) using simplex and multiplex PCR. Results In the present study, 107 (69%; 61 E. coli and 46 Klebsiella spp.) of 155 bacterial strains tested were found harboring at least one qnr gene consisting of 74 (47.74%) qnrB, 73 (47.10%) qnrS and 4 (2.58%) qnrA. Of the 107 strains encoding qnr genes, 102, 96 and 52 carried CTX-M1, TEM and SHV type ESBL respectively. Conclusion This study identified quinolone resistance (qnr) gene in ESBL-producing E. coli and Klebsiella spp. in Togo. These finding which suggest a possible resistance to quinolone are of high interest for better management of patients and control of antimicrobial resistance in Togo. Electronic supplementary material The online version of this article (10.1186/s13756-019-0552-0) contains supplementary material, which is available to authorized users.

Background Frequency of extended-spectrum-β-lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-β-lactamase-producing Clinical isolates in Pakistan. Methods A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data. Results A total of 497/995 (50%) isolates including Escherichia coli 65% (n = 321), Klebsiella spp. 25% (n = 124) and Pseudomonas. 5% (n = 24), Enterobacter spp. 4% (n = 20) and Acinetobacter spp. 2% (n = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% (n = 278) followed by pus 20% (n = 99) and wound swab 6% (n = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% (n = 392). PCR revealed bla CTX − M (76%) gene followed by bla OXA (52%), bla TEM (28%) and bla SHV (21%) were most prevalent among ESBL-producers detected by CDST. bla CTX − M − 1(65%), bla OXA (78%) and bla TEM (57%) genes were carried on plasmids. Amplicon sequencing revealed bla CTX − M − 15 (75%), bla OXA − 1 (49%) and bla TEM − 1B (34%) and 21 (n = 28) isolates carried three genes in them. Conclusion Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding bla CTXM − 15 gene and narrow spectrum bla OXA − 1 and bla TEM − 1B were also prevalent.

Background Carbapenemase-producing Enterobacteriaceae (CPE) is an important pathogen in nosocomial infections; nevertheless, only a few studies regarding CPE infection and its epidemiological factors have been conducted in the Republic of Korea (ROK). We aimed to analyze the clinical, microbiological, and epidemiological characteristics of CPE bloodstream infections (BSIs) in the ROK. Methods This retrospective cohort study included data collected from the National Surveillance System from January 2015 to December 2016 based on the epidemiologic survey performed by an epidemiologist from the Korea Centers for Disease Control and Prevention. We selected patients with CPE BSI from the Korea National Institute of Health based on carbapenemase genotyping. Results In this study, 131 CPE BSIs were identified, and the proportion of CPE BSI among total CPE isolates was 7%. Klebsiella pneumoniae accounted for 69% of all CPE BSIs, and 66% of these produced K. pneumoniae carbapenemase. Among nine provinces in ROK, one province had NDM as the most common carbapenemase. CPE was susceptible to amikacin, tigecycline, and gentamicin (76, 41, and 39%, respectively). Of 29 patients tested for colistin sensitivity, one patient showed colistin resistance. The most common CPE BSI sources were pneumonia, primary bacteremia, and biliary tract infection. Multivariable analysis showed that adequate antibiotic use at CPE detection was significantly associated with decreased 30-day mortality. Conclusions CPE BSIs are prevalent in the ROK. Moreover, most CPE BSIs originated from hospital-acquired infection, demonstrating the need to improve hospital infection control strategies. Electronic supplementary material The online version of this article (10.1186/s13756-019-0497-3) contains supplementary material, which is available to authorized users.

Background The problem of inappropriate use of antibiotics and the resulting growth in antimicrobial resistance (AMR) has implications for Poland and the world. The objective of this paper was to compare and contrast antibiotic resistance and antibiotic utilisation in Poland in recent years versus other European countries, including agreed quality indicators, alongside current AMR patterns and ongoing policies and initiatives in Poland to influence and improve antibiotic prescribing. Methods A quantitative ten-year analysis (2007–2016) of the use of antibiotics based on European Centre for Disease Prevention and Control (ECDC) data combined with a literature review on AMR rates and antimicrobial stewardship initiatives. Results The system of monitoring AMR and appropriate strategies to address AMR rates remain underdeveloped in Poland. The role of microbiological diagnostics and efforts to prevent infections is currently underestimated by physicians. Overall, Poland had one of the highest rates of total consumption of antibiotics in the analysed European countries. Total consumption of antibacterials for systemic use and relative consumption of beta-lactamase sensitive penicillins were characterized by small but statistically significant average annual increases between 2007 and 2016 (from 22.2 DIDs to 23.9 DIDs and from 0.8 to 1.3%, respectively). Conclusions The integrated activities around appropriate antibiotic prescribing in the pre- and post-graduate training of physicians and dentists seem to be particularly important, as well as changes in policies on prescribing antibiotics within ambulatory care. AMR and appropriate prescribing of antibiotics should be the focus of health policy actions in Poland.

Background Gram-negative ESKAPE bacteria are increasingly implicated in several difficult-to-treat infections in developed and developing countries. They are listed by the World Health Organization as resistant bacteria of critical priority in research. Objectives To determine the risk factors, prevalence, phenotypic profiles, genetic diversity and clonal relatedness of extended-spectrum β-lactamase (ESBL)-producing multi-drug resistant (MDR) Gram-negative ESKAPE bacteria in the faecal carriage and clinical samples from patients in an urban, tertiary and a rural, district hospital in uMgungundlovu District, KwaZulu-Natal, South Africa. Methods This study took place in a district and tertiary hospital during a two-months period from May to June 2017 in uMgungundlovu district, South Africa. Rectal swabs collected from hospitalized patients, at admission, after 48 h and at discharge (whenever possible) formed the carriage sample while clinical isolates routinely processed in the microbiological laboratory during the sampling period were also collected and formed the clinical sample. Gram-negative ESKAPE bacteria were screened for ESBL production on selective MacConkey agar and confirmed using ROSCO kits. Minimum inhibitory concentrations were determined, and real-time and multiplex polymerase chain reaction were used to ascertain the presence of blaCTX-M group-1-2-9, blaCTX-M group 8/25, blaSHV, blaTEM, blaOXA-1-like, blaKPC, blaVIM, blaIMP, blaGES and AmpC genes. Genomic fingerprinting was also performed using ERIC-PCR. Risk factors for ESBL-mediating MDR Gram-negative ESKAPE colonization were ascertained by univariate and multivariate logistic regression analyses. Results Overall prevalence of carriage of ESBL-mediating MDR Gram-negative ESKAPE was 37.21% (16/43), 42.31% (11/26) and 57.14% (4/7) at admission, after 48 h and at discharge respectively. The prevalence of ESBL-mediating MDR Gram-negative ESKAPE bacteria in faecal carriage (46%) was higher than clinical samples (28%). Colonization was mainly associated with the referral from district to tertiary hospital with high statistical significance (OR: 14.40, 95% CI 0.98–210.84). blaCTX-M-group-9, blaCTX-M-group-1 and blaSHV were the main resistance genes identified. Several patients carried more than two different isolates. A Klebsiella pneumoniae (K1) clone was circulating within wards and between hospitals. Conclusion The study highlights the high prevalence of ESBL-mediating MDR Gram-negative ESKAPE bacteria in carriage and clinical samples among hospitalized patients in uMgungundlovu, South Africa. The wide dissemination of these resistant ESKAPE bacteria in hospitals necessitates improvements in routine screening and reinforcement of infection, prevention and control measures. Electronic supplementary material The online version of this article (10.1186/s13756-018-0423-0) contains supplementary material, which is available to authorized users.

Background Metallo-β-lactamase (MBL)-producing isolates have a strong impact on diagnostic and therapeutic decisions. A high frequency of MBL-producing gram-negative bacilli has been reported worldwide. The current study was based on determining the incidence of MBL-producing imipenem-resistant clinical isolates and investigating the β-lactamase gene variants in strains conferring resistance to a carbapenem drug (imipenem). Methods A total of 924 gram negative isolates were recovered from a tertiary care hospital in Lahore, Pakistan, during a two-year period (July 2015 to February 2017). The initial selection of bacterial isolates was based on antibiotic susceptibility testing. Strains resistant to imipenem were processed for the molecular screening of β-lactamase genes. Statistical analysis for risk factor determination was based on age, gender, clinical specimen and type of infection. Results The rate of imipenem resistance was calculated to be 56.51%. Among the 142 strains processed, the phenotypic tests revealed that the incidence of MBLs was 63.38% and 86.61% based on the combination disc test and the modified Hodge test, respectively. The frequencies of bla TEM, bla SHV, bla OXA, bla IMP-1, and bla VIM genes were calculated to be 46%, 34%, 24%, 12.5% and 7%, respectively. The co-expression of bla MBL (bla IMP and bla VIM) and bla ESBL (bla TEM, bla SHV, bla OXA) was also detected through multiplex and singleplex PCR. bla OXA, bla TEM and bla SHV coexisted in 82% of the isolates. Co-expression of ESBL and MBL genes was found in 7% of the isolates. Conclusion To our knowledge, this is the first report from Pakistan presenting the concomitant expression of bla OXA, bla TEM and bla SHV with bla IMP-1 and bla VIM in MBL-producing gram-negative bacilli.

Background The emergence and spread of New Delhi metallo-β-lactamase-producing Enterobacteriaceae has been a serious challenge to manage in the clinic due to its rapid dissemination of multi-drug resistance worldwide. As one main type of carbapenemases, New Delhi metallo-β-lactamase (NDM)is able to confer resistance to almost all β-lactams, including carbapenems, in Enterobacteriaceae. Recently, New Delhi metallo-β-lactamase-5 attracted extensive attention because of increased resistance to carbapenems and widespread dissemination. However, the dissemination mechanism of blaNDM-5 gene remains unclear. Methods A total of 224 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected from different hospitals in Zhejiang province. NDM-5-positive isolates were identified and subjected to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of blaNDM-5 with southern blot hybridisation, and analysed plasmids containing blaNDM-5 with filter mating and DNA sequencing. Results Eleven New Delhi metallo-β-lactamase-5 (NDM-5)-producing strains were identified, including 9 Escherichia coli strains, 1 Klebsiella pneumoniae strain, and 1 Citrobacter freundii strain. No epidemiological links for E. coli isolates were identified by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). S1-PFGE and southern blot suggested that the blaNDM-5 gene was located on a 46-kb IncX3-type plasmid in all isolates. Nine of the 11 isolates (81.8%) tested could successfully transfer their carbapenem-resistant phenotype to E. coli strain C600. Moreover, sequence analysis further showed that this plasmid possessed high sequence similarity to most of previously reported blaNDM-5-habouring plasmids in China. Conclusion The present data in this study showed the IncX3 type plasmid played an important role in the dissemination of blaNDM-5 in Enterobacteriaceae. In addition, to the best of our knowledge, this report is the first to isolate both E. coli and C. freundii strains carrying blaNDM-5 from one single patient, which further indicated the possibility of blaNDM-5 transmission among diverse species. Close surveillance is urgently needed to monitor the further dissemination of NDM-5-producing isolates. Electronic supplementary material The online version of this article (10.1186/s13756-018-0349-6) contains supplementary material, which is available to authorized users.

Background Antibiotic resistance of environmental Escherichia coli in hospitals could be increased due to extensive use of biocides resulting in serious infections. In this study, the prevalence of antibiotic resistance of environmental isolates of E. coli from hospitals and household settings were evaluated and compared. In addition, the association between biocide minimum inhibitory concentration (MIC) and multiple drug resistance (MDR) was investigated. Methods Environmental samples were collected from different homes and hospitals in Amman, Jordan. The isolates were identified phenotypically and by PCR. Antibiotic susceptibility tests and MIC of selected biocides were performed on the isolates. Screening for blaCTX-M group 1 was also performed. Results Of 21 E. coli strains isolated, 47.6% were MDR and 67.9% were phenotypically identified as extended spectrum beta-lactamase (ESBL) producers. The occurrence of these ESBL isolates was comparable between household and hospital settings (P > 0.05). The MIC values of the biocides tested against all isolates were well below the in-use concentration of biocides. Moreover, the MICs of biocides were comparable between isolates from households and those from hospitals (P > 0.05). No association was found between MDR and biocide MIC (P > 0.05). Most of ESBL isolates harboured blaCTX-M 1. Conclusions The extensive use of biocides in hospitals is not associated with MDR nor does it affect the MIC of biocides against E.coli.