Background: The emergence and worldwide circulation of strains of beta-lactamase-producing Enterobacterales represent a major public health problem. The aim of this study was the characterization of extended spectrum beta-lactamase (ESBL) and carbapenemase-producing strains of Escherichia coli and Klebsiella pneumoniae resistant strains isolated in Kinshasa hospitals. Methods: Urine samples were collected from hospitalized and non-hospitalized patients. Bacteria were identified by using conventional biochemical tests and MALDI-TOF. Antibiotic susceptibility testing was performed by disk diffusion method. ESBL and carbapenemase phenotypes were screened using standard microbiological tests. ESBL-encoding genes were tested by multiplex end-point PCR assays and carbapenemase enzymes were identified by immunochromatography assay. Results: Out of 286 urines, 200 (69.9%) Gram negative bacteria were isolated, among them 70 (35.0 %) E. coli and 23 (11.5%) K. pneumoniae. ESBL were identified in 62.9 % (44/70) and in 52.2% (12/23) of E. coli and K. pneumoniae strains respectively. The rate of ESBL-producing Enterobacterales was 60.2% (56/93). Among ESBL producers, the blaCTX-M1 gene was identified in 88.6 % (39/44) isolates of E. coli and in 100% (12/12) isolates of K. pneumoniae. The bla-gene blaCTX-M-1 was identified in 91.2% (51/56) of all ESBL producers strains. Only 3.2% (3/93) of Enterobacterales strains produced the NDM enzyme. The Other genes encoding beta-lactamase were blaTEM (64.3 %), blaOXA-1/-30 (58.9 %), and blaSHV (39.3%). Conclusion: A high rate of ESBL-producing Enterobacterales was observed. The results of the study report the first description of metallo-β-lactamase NDM producing E. coli and K. pneumoniae strains from patients in Democratic Republic of Congo.

Abstract Background Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. Methods 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). Results According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). Conclusions The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Objectives: This study evaluated the performance of the Novodiag® CarbaR+ an automated qualitative nucleic acid-based diagnostic assay detecting the blaVIM, blaNDM, blaIMP, blaOXA-48, blaKPC, blaOXA-23, blaOXA-58, blaOXA-24, and ISAba1 associated blaOXA-51 carbapenemase genes and colistin resistance mcr-1 gene from clinical isolates or directly from rectal swabs. Materials and Methods: CarbaR+ was evaluated on 201 clinical isolates and on 100 rectal swabs (80 selected swabs from patients that were evaluated by culture method and/or Xpert Carba-R assay and 20 spiked samples). PCR-sequencing on colonies was considered as the gold standard. Results: The CarbaR+ detected all the variants of the targeted resistance genes (39 blaVIM-, 30 blaNDM-, 20 blaIMP-, 19 blaOXA-48-, 15 blaKPC-, 19 blaOXA-23-, 13 blaOXA-58-, 4 blaOXA-24-, 8 ISAba1-blaOXA-51-, and 3 mcr-1-like genes) with sensitivity and specificity of 98.2% and 99.7%, respectively. On the 80 rectal swabs, 71 CarbaR+ results were fully concordant with the results on selective culture media (66 positive samples with 1 to 3 carbapenemases and 5 negative samples). In eight rectal swabs, CarbaR+ identified additional carbapenemase genes. One false negative result with an Escherichia coli producing-OXA-181 was observed and one CarbaR+ result for OXA-48 was in agreement with Xpert Carba-R assay, without growth on culture media. A concordance of 100% was observed on spiked samples. Conclusions: Novodiag CarbaR+ is a random-access fully automated system that achieves excellent performances for the detection of carbapenemase and/or colistin resistance determinants either from cultured clinical isolates or directly from rectal swabs in 80 minutes.

Objectives: To describe a novel plasmid‐borne class D carbapenemase (CHDL) named OXA‐427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital. Methods: OXA‐427‐producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA‐427‐encoding plasmid and cloning). The antimicrobial resistance profile of OXA‐427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53. Results: Eleven OXA‐427‐producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA‐427 shares only 22%‐29% amino acid identity with OXA‐48‐like enzymes and other acquired CHDL (e.g. OXA‐23, ‐24/40 and ‐58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D &bgr;‐lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA‐427 hydrolysed imipenem and conferred resistance to extended‐spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA‐427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid. Conclusions: OXA‐427 is a novel CHDL most closely related to chromosomal class D &bgr;‐lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA‐427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period.

Four screening assays aimed for rapid detection of carbapenemase production from Gram-negative bacterial isolates, i.e., the Neo-Rapid Carb kit (Rosco Diagnostica A/S), the Rapidec Carba NP test (bioMérieux SA), the β Carba test (Bio-Rad Laboratories N.V.), and a homemade electrochemical assay (BYG Carba test) were evaluated against a panel comprising 328 clinical isolates (Enterobacteriaceae [n = 198] and nonfermentative Gram-negative bacilli [n = 130]) with previously characterized resistance mechanisms to carbapenems. Among Enterobacteriaceae isolates, the BYG Carba test and the β Carba test showed excellent sensitivities (respectively, 100% and 97.3%) and specificities (respectively, 98.9% and 97.7%). The two other assays yielded poorer performances with sensitivity and specificity of 91.9% and 83.9% for the Rapidec Carba NP test and of 89.2% and 89.7% for the Neo-Rapid Carb kit, respectively. Among Pseudomonas spp., sensitivities and specificities ranged, respectively, from 87.3% to 92.7% and from 88.2% to 94.1%. Finally, all tests performed poorly against Acinetobacter spp., with sensitivities and specificities, respectively, ranging from 27.3% to 75.8% and from 75 to 100%. Among commercially available assays, the β Carba test appeared to be the most convenient for routine use and showed the best overall performances, especially against OXA-48-like producers. The excellent performance of the BYG Carba test against Enterobacteriaceae was confirmed (100% sensitivity and 98.9% specificity).

The study aimed to characterize beta-lactam resistance mechanisms of Enterobacteriaceae isolates recovered from diseased dogs and cats between 2008 and 2010 in a European surveillance program (ComPath I) for the antibiotic susceptibility of bacterial pathogens. A total of 608 non-duplicated Enterobacteriaceae isolates were obtained prior antibiotic treatment from diseased dogs (n=464) and cats (n=144). Among the 608 Enterobacteriaceae isolates, 22 presented a minimal inhibitory concentration against cefotaxime above EUCAST breakpoints of susceptibility. All the 22 isolates remained susceptible to carbapenems. Ten isolates were confirmed as extended-spectrum-beta-lactamase (ESBL) producers by PCR-sequencing of bla coding genes including 9 blaCTX-M (CTX-M-1, 14, 15, 32,…) and 1 blaTEM-52 and 12 were AmpC-producing isolates (10 plasmidic CMY-2 group and 2 isolates overexpressing their chromosomal AmpC). ESBLs and plasmid-mediated AmpC (pAmpC)-producing isolates were mainly recovered from dogs (n=17) suffering from urinary tract infections (n=13) and originated from eight different countries. ESBL-bearing plasmids were mostly associated with IncFII incompatibility groups while CMY-2 was predominantly associated with plasmid of the IncI1 group. ESBL/pAmpC-producing Escherichia coli belonged to phylogroup A (n=5), B2 (n=4), and D (n=5). Multilocus sequence typing analysis revealed that among three CTX-M-15-producing E. coli, two belong to sequence type (ST) 131 and one to ST405. The presence of CTX-M-15 including on IncFII plasmids in E. coli ST131-B2 has also been described in isolates of human origin. This suggests the possibility of exchanges of these isolates from humans to companion animals or vice-versa.

During a polymerase chain reaction (PCR)-based surveillance study of β-lactam resistance, 19 OXA-48-positive enterobacterial isolates were detected at nine Belgian hospitals from January 2010 to April 2011. Most cases were presumed to have been locally acquired and were detected in patients who had not travelled abroad. Clonally related outbreaks occurred in two different cities. The majority of isolates co-produced several β-lactamases as well as non-β-lactam resistance genes. This report highlights the rapid emergence and spread of OXA-48-producing Enterobacteriaceae in Belgium.

ABSTRACT We report the first description of the metallo-β-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. bla VIM-31 was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested beta-lactam agents (except for ertapenem), resulting from lower k cat (except for ertapenem) and higher K m values for VIM-31.

IMP-13 is a class B metallo-β-lactamase that was first described in a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa from Italy.1 The IMP-13 gene was mostly described as part of the class 1 InPSG integron located on a Tn5051 transposon, and it was reported in intercontinentally disseminating P. aeruginosa of sequence type 621 causing outbreaks in Italy, Romania and Argentina.1–3 Multidrug-resistant P. aeruginosa are at the origin of outbreaks that are often difficult to control and to eradicate. The persistence of pools of transferable elements carrying antimicrobial resistance genes in hidden environmental reservoirs may further explain the resurgence of some epidemics, as well as their ability to quickly appear. The role of Pseudomonas putida as a metallo-β-lactamase reservoir was recently reinforced by showing its ability to transfer carbapenem resistance to P. aeruginosa.4 We briefly describe here the carriage of blaIMP-13 in Pseudomonas monteilii, a species closely related to P. putida, recovered from the clinical environment.

Sir, IMP-13 is a class B metallo-b-lactamase that was first described in a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa from Italy. The IMP-13 gene was mostly described as part of the class 1 InPSG integron located on a Tn5051 transposon, and it was reported in intercontinentally disseminating P. aeruginosa of sequence type 621 causing outbreaks in Italy, Romania and Argentina. – 3 Multidrug-resistant P. aeruginosa are at the origin of outbreaks that are often difficult to control and to eradicate. The persistence of pools of transferable elements carrying antimicrobial resistance genes in hidden environmental reservoirs may further explain the resurgence of some epidemics, as well as their ability to quickly appear. The role of Pseudomonas putida as a metallo-b-lactamase reservoir was recently reinforced by showing its ability to transfer carbapenem resistance to P. aeruginosa. We briefly describe here the carriage of blaIMP-13 in Pseudomonas monteilii, a species closely related to P. putida, recovered from the clinical environment. In the setting of an outbreak due to a GES-12-producing Acinetobacter baumannii occurring in 2009, the inanimate environment of an acute care burn unit of a Belgian hospital was regularly screened. In January 2011, the materials and surfaces in multiple hospital environments were sampled. In total, .70 different specimens were obtained from both dry inanimate surfaces (floor, bed frame, bed linen and computer pads) and wet surroundings (sinks, baths, toilets, toilet brushes and soap). This screening was carried out all over the burn unit as well as in the operating rooms, corridors and nurse restrooms. Screening cultures were grown overnight at 358C on a MacConkey plate onto which a 30 mg ceftazidime disc (Bio-Rad, Nazareth, Belgium) was placed. While no A. baumannii could be recovered from any of the specimens, culture of a toilet brush sample yielded the growth of a ceftazidime-resistant isolate that was presumptively identified as P. putida by Vitek 2 GN-ID cards. However, this isolate was subsequently confirmed as P. monteilii both by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (Microflex LT, Bruker Daltonics, Leipzig, Germany) and by sequencing of the gene encoding 16S rRNA (100% identity with P. monteilii GAPP1, accession no. GU396289), emphasizing the difficulty of correctly distinguishing between P. putida and P. monteilii by conventional biochemical methods. Antimicrobial susceptibility testing by agar disc diffusion according to CLSI guidelines revealed a multidrugand carbapenem-resistant isolate, which only remained susceptible to colistin and intermediately susceptible to aztreonam. MICs, determined according to the CLSI microdilution reference method, confirmed the highlevel resistance of this environmental isolate to extendedspectrum b-lactamases (ESBLs; MICs of ceftazidime and cefepime .128 mg/L, and MICs of imipenem and meropenem .32 mg/L). Double discs showed the presence of a synergy between imipenem (10 mg) and EDTA (465 mg; 10 mL of 0.125 M EDTA stock), which highly suggested the presence of a metallo-b-lactamase. PCR sequencing confirmed the presence of a blaIMP-13 gene located upstream of a mutated gene encoding an aminoglycoside-modifying enzyme, aacA4-2, embedded in a new class I integron, In449, which is slightly different (six mutations in 1889 nucleotides analysed; accession number JN091097) from the InPSG already described in P. aeruginosa. Moreover, PCR performed with primers specific to the transposase (tnpA) and the resolvase (tnpR) of Tn5051 were negative, indicating that this integron is located on a different and unknown genetic structure. Neither genes encoding ESBLs (blaSHV, BEL, VEB, PER and GES) nor oxacillinase of groups 1, 2 or 10 could be detected by PCR. Plasmid extractions performed using the Kieser method or with Qiagen midi preparation did not yield any plasmidic bands and several attempts to obtain IMP-13-expressing electroporants in PA01 wild-type P. aeruginosa failed, suggesting the possible location of blaIMP-13 on the bacterial chromosome. Subsequent to this screening, reinforced infection control measures were taken, including discarding of all toilet brushes, reviewing and extensive explanation to the housekeeping personnel of the standard operation cleaning/disinfection procedures, implementation of antibacterial filters on taps, reinforced practice of hand hygiene disinfection, daily cleaning of the entire unit, systematic environmental screening of infected/colonized patients’ rooms upon discharge, enhanced

ABSTRACT Five multidrug-resistant nonclonally related Enterobacteriaceae isolates were recovered in Belgium in 2010 from three patients who had been hospitalized in Pakistan, Montenegro, and Serbia/Kosovo. New Delhi metallo-β-lactamase (NDM-1) was detected in each of the isolates in addition to several extended-spectrum β-lactamases (CTX-M-15, SHV-12), plasmidic cephalosporinases (CMY-16, CMY-58), rRNA methylases (ArmA, RmtB), and Qnr genes (qnrA6, qnrB1, qnrB2). One patient died from uncontrolled sepsis, while the two others recovered. No secondary cases occurred in any of the hospitals.