Phenotypic identification of different ß-Lactamases in intrinsic and acquired colistin resistant uropathogenic gram negative bacteria.

Objective: Identification of MBL, AmpC and ESBLs in colistin intrinsic and acquired resistant uropathogenic gram negative bacteria. Method: Urine samples were collected from Hayatabad Medical Complex, Peshawar during 17 January to 30 June 2019. Collected urine samples were aseptically transported microbiology lab of Health Research Institution (HRI), National Institute of Health (NIH), Khyber Medical College, Peshawar and streaked on different media. Positive growth was identified by API-10s. Antibiotic sensitivity profile was done by Modified Kirby Bauer disc diffusion method. Detection of metallo ßlactamases (MBL) production by Imipenem EDTA synergy test, Double Disc Synergy Test (DDST) for detection of ESBLs and D-test for the detection of inducible AmpC beta lactamases test was used. Colistin resistance was identified via broth micro dilution according to CLSI manual. Colistin resistant bacteria was divided in two categories; acquired and intrinsic resistant bacteria according to CLSI manual. Results: Out of 2000 urine samples, 281(14%) gram-negative bacteria were isolated. Among positive samples, acquired colistin resistant bacteria were 241 and intrinsic resistant bacteria were 40 isolates. MBL was produce by twenty one (11.7%) E.coli and seventeen (40.5%) Pseudomonas aeruginosa. E. coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Serratia Oderifora and Proteus Marblis were ESBLs producing bacteria. AmpC production was prevalent in fourteen (7.8%) E. coli and twelve (28.6%) Pseudomonas aeruginosa. Fifty-five samples showed resistance to colistin out of 241 samples. In colistin resistant bacteria, two E.coli were MBL, ESBLs, while one E.coli was ESBLs, AmpC co-producing bacteria. The most prevalent extended drug resistant bacteria were Pseudomonas aeruginosa (28.6%) and Escherichia coli (6.1%), While 155(86.6%) Escherichia coli, 25 (59.5%) Pseudomonas aeruginosa and 22 (95.7%) Serratia Oderifora was multi drug resistant bacteria. Conclusion: Current study concluded that ESBL, MBL AmpC enzymes and their co-expression was observed with colistin resistance in E.coli and Pseudomonas aeruginosa.

Whole-Genome Analysis of Extensively Drug-Resistant Enterobacter hormaechei Isolated from a Patient with Non-Hodgkin's Lymphoma.

BACKGROUND: Currently, the Enterobacteriaceae species are responsible for a variety of serious infections and are already considered a global public health problem, especially in underdeveloped countries, where surveillance and monitoring programs are still scarce and limited. Analyses were performed on the complete genome of an extensively antibiotic-resistant strain of Enterobater hormaechei, which was isolated from a patient with non-Hodgkin's lymphoma, who had been admitted to a hospital in the city of Manaus, Brazil. METHODS: Phenotypical identification and susceptibility tests were performed in automated equipment. Total DNA extraction was performed using the PureLink genomic DNA mini-Kit. The genomic DNA library was prepared with Illumina Microbial Amplicon Prep and sequenced in the MiSeq Illumina Platform. The assembly of the whole-genome and individual analyses of specific resistance genes extracted were carried out using online tools and the Geneious Prime software. RESULTS: The analyses identified an extensively resistant ST90 clone of E. hormaechei carrying different genes, including blaCTX-M-15, blaGES-2, blaTEM-1A, blaACT-15, blaOXA-1 and blaNDM-1, [aac(3)-IIa, aac(6')-Ian, ant(2″)-Ia], [aac(6')-Ib-cr, (qnrB1)], dfrA25, sul1 and sul2, catB3, fosA, and qnrB, in addition to resistance to chlorhexidine, which is widely used in patient antisepsis. CONCLUSIONS: These findings highlight the need for actions to control and monitor these pathogens in the hospital environment.

Piperacillin/tazobactam Susceptibility Test Interpretive Criteria for Enterobacterales: Recommendations from the United States Committee on Antimicrobial Susceptibility Testing.

The in vitro susceptibility testing interpretive criteria (STIC) for TZP against Enterobacterales were recently updated by the Food and Drug Administration (FDA), Clinical & Laboratory Standards Institute (CLSI), and European Committee on Antimicrobial Susceptibility Testing (EUCAST). The United States Committee on Antimicrobial Susceptibility Testing (USCAST) also recently reviewed TZP STIC for Enterobacterales and arrived at different STIC for Enterobacterales and herein we explain our recommendations and rationale behind them. Based on our review of the available data, USCAST does not recommend TZP STIC for certain Enterobacterales species that have a moderate to high likelihood of clinically significant AmpC production (E. cloacae, C. freundii, and K. aerogenes only) or for third-generation cephalosporin-non-susceptible (3GC-NS) Enterobacterales. USCAST recommends a TZP susceptibility breakpoint of ≤ 16/4 mg/L for third-generation cephalosporin-susceptible (3GC-S) Enterobacterales but only endorses the use of extended infusion TZP regimens for patients with infections due to these pathogens.

Impact of extended-spectrum chromosomal AmpC (ESAC) of Escherichia coli on susceptibility to cefiderocol.

The impact of chromosomally encoded wild-type or extended-spectrum (ESAC) AmpC ß-lactamases of Escherichia coli on susceptibility to ceftazidime, cefepime, and cefiderocol was evaluated in different genetic backgrounds, including wild-type, PBP3-modified, and porin-deficient E. coli strains. Recombinant E. coli strains possessing the different backgrounds and producing variable ESACs were evaluated. Although ESAC enzymes conferred resistance to ceftazidime and decreased susceptibility to cefepime as expected, we showed here that cefiderocol was also a substrate of ESAC enzymes. IMPORTANCE: We showed here that chromosomally encoded intrinsic extended-spectrum cephalosporinases of Escherichia coli may impact susceptibility not only to ceftazidime and cefepime but also to cefiderocol.

Characterisation of a novel AmpC beta-lactamase, DHA-33, resistant to inhibition by cloxacillin.

Plasmid-encoded DHA-type AmpCs have been extensively reported in Enterobacterales. The expression of the genes encoding these plasmid-mediated enzymes are inducible and these enzymes are capable of conferring resistance to a wide spectrum of beta-lactams including penicillins and broad-spectrum cephalosporins. The identification of infections caused by AmpC-producing bacteria is a necessity, both for infection control/epidemiology purposes and to inform treatment choices. A common testing method for AmpC production in the clinical laboratory setting is to supplement Mueller-Hinton agar plates used for antibiotic disk diffusion with cloxacillin, a potent inhibitor of AmpC enzymes. Here we describe a novel DHA variant, produced by a clinical Escherichia coli isolate, which is resistant to cloxacillin inhibition.

The resistance patterns and molecular characteristics of ESBL/AmpC-producing Escherichia coli from captive panda ecosystem in China.

Escherichia coli (E. coli) plays an important ecological role, and is a useful bioindicator to recognize the evolution of resistance in human, animal and environment. Recently, extended-spectrum ß-lactamases (ESBL) producing E.coli has posed a threat to public health. Generally, captive healthy giant pandas are not exposed to antibiotics; however, they still acquire antimicrobial resistant bacteria. In order to understand whether there is an exchange of resistance genes within the ecosystems of captive giant pandas, this study explored resistance characteristics of 330 commensal E. coli isolates from feces of giant pandas, the surroundings, and breeders. Isolates from different sources showed similar resistance phenotype, and ESBL/AmpC-producing isolates showed more profound resistance to antibiotics than non-ESBL/AmpC-producing isolates (P<0.05). Furthermore, the occurrence of broad-spectrum ß-lactamase related resistance genes and colistin resistance genes was detected, and isolates phylogenetic typing and multilocus sequence typing (MLST) were applied in this study. Seven different ß-lactamase resistance genes (blaCTX-M-55, blaCTX-M-15, blaCTX-M-27, blaCTX-M-65, blaTEM-1, blaOXA-1 and blaCMY) and mcr-1 were found in 68 ESBL/AmpC-producing isolates. blaCTX-M-55 (48.53 %) was found the most predominant resistance genes, followed by blaTEM-1 (19.12 %) and blaCTX-M-27 (16.18 %). Nonetheless, blaCTX-M-55 was commonly detected in the isolates from giant pandas (63.16 %), the surroundings (43.48 %), and breeders (33.33 %). However, there were no carbapenemase genes detected in this study. mcr-1 was harbored in only one isolate from giant panda. Forty-five tansconjugants were successfully obtained in the conjugation experiments. The presence of antimicrobial resistance and related resistance genes tested were observed in the transconjugants. The results indicated that 52.63 % of the isolates from giant panda 73.91 % of the isolates from surroundings, and 100 % of the isolates from breeders were phylogroup A. Total of 27 sequence types (ST) were recognized from the isolate by MLST and found that ST48 (19/68; 27.94 %) was the predominant ST type, especially in the isolates from giant pandas and the surroundings. In conclusion, commensal ESBL/AmpC-producing E. coli becomes a reservoir of ESBL resistance genes, which is a potential threaten to health of giant pandas. The interaction between giant pandas, surroundings and breeders contribute to development of resistant phenotypes and genotypes which might transfer across species or the surroundings easily; hence, strict monitoring based on a "One Health" approach is recommended.

MAST® D72C test: a novel option for ESBL, AmpC and carbapenemase detection.

PURPOSE: The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. METHOD: The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in ß-lactamases, and compared with that of the reference double disk synergy test. ß-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several ß-lactamases, and 22 strains that produced other ß-lactamases. RESULTS: The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. CONCLUSION: These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories.

A Case of Infective Endocarditis Caused by Citrobacter koseri: Unraveling a Rare Pathogen and Dire Outcome.

Citrobacter koseri (formerly classified as Citrobacter diversus) is a gram-negative bacillus (GNB) that occurs as an opportunistic pathogen in neonates and immunocompromised patients. Citrobacter species have been implicated in nosocomial settings leading to infections involving the urinary tract, respiratory tract, liver, biliary tract, meninges, and even in rarer conditions-blood stream infection and infective endocarditis (IE). Gram-negative bacilli are responsible for 3% to 4% of all IE cases and have been traditionally associated with intravenous drug users. Patients with non-HACEK (species other than Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, or Kinglella species) GNB IE have poor clinical outcomes with higher rates of in-hospital mortality and complications. The American Heart Association (AHA) and Infectious Diseases Society of America (IDSA) both recommend the use of combination antibiotic therapy with a beta-lactam (penicillins, cephalosporins, or carbapenems) and either an aminoglycoside or fluoroquinolones for 6 weeks (about 1 and a half months) to treat IE due to non-HACEK GNB. Citrobacter koseri is becoming more recognized due to its inherent resistance to ampicillin and emerging drug resistance to beta lactams and aminoglycosides requiring carbapenem therapy. Our case is of a 75-year-old male with no previously reported history of primary or secondary immunodeficiency disorders who developed C koseri blood stream infection. His infectious work-up revealed mitral valve IE and septic cerebral emboli resulting in ischemic infarcts. This case illustrates the importance of recognizing GNB organisms as rising human pathogens in IE cases even without active injection drug use or nosocomial exposure.

Complete genome sequence of Yokenella regensburgei isolated from a patient with urinary tract infection in India.

Yokenella regensburgei, an environmental organism, is an emerging pathogen in patients chiefly with immune suppression. We report the draft genome of Y. regensburgei, strain UU2206353, isolated from the urinary tract of an immunocompetent individual. The assembled genome consisted of 4,669,536 bp distributed over 20 contigs with 4,283 protein-coding genes.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type ß-lactamase.

INTRODUCTION: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. METHODS: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. RESULTS: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. CONCLUSIONS: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.

Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids.

BACKGROUND: Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. METHODS: In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n = 192 ESC-R E. coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. FINDINGS: We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E. coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E. coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. INTERPRETATION: We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. FUNDING: This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

What Contributes to the MIC? Beyond ß-Lactamase Gene Detection in Klebsiella pneumoniae.

BACKGROUND: K. pneumoniae is capable of resistance to ß-lactam antibiotics through expression of ß-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which ß-lactamases and outer membrane porins affected ß-lactam resistance. METHODS: MICs to ß-lactams and inhibitor combinations were determined by agar dilution or E-test. Outer membrane porin production was evaluated by western blot of outer membrane fractions. ß-lactamase carriage was determined by whole genome sequencing and expression evaluated by RT-qPCR. RESULTS: Plasmid-encoded ß--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane/tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. ESßLs and pAmpCs in addition to porin loss were sufficient to confer resistance to the third generation cephalosporins, pipercillin/tazobactam, ceftolozane/tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to pipercillin/tazobactam. DISCUSSION: Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. some ß-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal ß-lactamases and loss of porins.

Research on Intelligent Vehicle Trajectory Tracking Control Based on Improved Adaptive MPC.

Intelligent vehicle trajectory tracking exhibits problems such as low adaptability, low tracking accuracy, and poor robustness in complex driving environments with uncertain road conditions. Therefore, an improved method of adaptive model predictive control (AMPC) for trajectory tracking was designed in this study to increase the corresponding tracking accuracy and driving stability of intelligent vehicles under uncertain and complex working conditions. First, based on the unscented Kalman filter, longitudinal speed, yaw speed, and lateral acceleration were considered as the observed variables of the measurement equation to estimate the lateral force of the front and rear tires accurately in real time. Subsequently, an adaptive correction estimation strategy for tire cornering stiffness was designed, an AMPC method was established, and a dynamic prediction time-domain adaptive model was constructed for optimization according to vehicle speed and road adhesion conditions. The improved AMPC method for trajectory tracking was then realized. Finally, the control effectiveness and trajectory tracking accuracy of the proposed AMPC technique were verified via co-simulation using CarSim and MATLAB/Simulink. From the results, a low lateral position error and heading angle error in trajectory tracking were obtained under different vehicle driving conditions and road adhesion conditions, producing high trajectory-tracking control accuracy. Thus, this work provides an important reference for improving the adaptability, robustness, and optimization of intelligent vehicle tracking control systems.

Antimicrobial activity of cefepime-tazobactam combination against extended spectrum beta-lactamase and/or AmpC beta-lactamase- producing gram-negative bacilli.

BACKGROUND: The problem of resistance to beta-lactam antibiotics, which is caused by ESBL and AmpC ß-lactamases, is getting worse globally. Infections caused by bacterial isolates harboring these enzymes are difficult to treat with carbapenems being the sole effective treatment option for such infections. The objective of this study was to determine the frequency of ESBLs and AmpC-producing Gram-negative bacilli isolated from clinical specimens and to evaluate the sensitivity of cefepime-tazobactam combination against them. METHODS: This is an observational cross-sectional study carried out on 100 Gram-negative bacilli at Theodor Bilharz Research Institute Hospital during the period from February 2015 to January 2016. ESBL production was screened by using the disc diffusion test followed by confirmation by the combined disc confirmatory test, the screening for AmpC production was conducted using the cefoxitin disc test, which was subsequently confirmed by the AmpC disc test. Isolates confirmed positive for ESBL and/ or AmpC production were investigated for their susceptibility to antibiotics. RESULTS: Among 100 Gram-negative bacilli, 44 isolates were confirmed as ESBL producers by the combined disc confirmatory test out of 56 isolates that tested positive for ESBL production through the disc diffusion test. The presence of AmpC production was assessed using the cefoxitin disc test, 32 isolates were screened to be AmpC producers, and the AmpC disc test confirmed AmpC production in 9 isolates of them. Using the Mast® D68C set, 32 isolates were ESBL producers, 3 were AmpC producers, and 4 isolates were ESBL/AmpC co-producers. The highest sensitivity was to cefepime-tazobactam (91.48%) followed by the carbapenems. CONCLUSION: Cefepime-tazobactam showed remarkable activity against ESBL and/or AmpC-producing Gram-negative bacilli and may be considered as a therapeutic alternative to carbapenems.

Upregulation of pmrA, pmrB, pmrC, phoQ, phoP, and arnT genes contributing to resistance to colistin in superbug Klebsiella pneumoniae isolates from human clinical samples in Tehran, Iran.

Background: Antibiotic resistance in Klebsiella pneumoniae isolates, particularly resistance to colistin, has become a growing concern. This study seeks to investigate the upregulation of specific genes (pmrA, pmrB, pmrC, phoQ, phoP, and arnT) that contribute to colistin resistance in K. pneumoniae isolates collected from human clinical samples in Tehran, Iran. Methods: Thirty eight K. pneumoniae isolates were obtained and subjected to antibiotic susceptibility testing, as well as evaluation for phenotypic AmpC and ESBL production according to CLSI guidelines. The investigation of antibiotic resistance genes was conducted using polymerase chain reaction (PCR), whereas the quantification of colistin resistance related genes expressions was performed via Real-Time PCR. Results: The highest and lowest antibiotics resistance were observed for cefotaxime 33 (86.8%) and minocycline 8 (21.1%), respectively. Twenty-four (63.2%) and 31 (81.6%) isolates carried AmpC and ESBLs, respectively. Also, antibiotic resistance genes containing blaNDM, blaIMP, blaVIM, blaSHV, blaTEM, blaCTXM, qnrA, qnrB, qnrS, and aac(6')-Ib were detected in K. pneumoniae isolates. Only 5 (13.1%) isolates were resistant to colistin and the MIC range of these isolates was between 4 and 64 µg ml-1. Upregulation of the pmrA, pmrB, pmrC, phoQ, phoP, and arnT genes was observed in colistin-resistant isolates. The colistin-resistant isolates were found to possess a simultaneous presence of ESBLs, AmpC, fluoroquinolone, aminoglycoside, and carbapenem resistant genes. Conclusions: This study reveals escalating antibiotic resistance in K. pneumoniae, with notable coexistence of various resistance traits, emphasizing the need for vigilant surveillance and innovative interventions.

The ICU environment contributes to the endemicity of the "Serratia marcescens complex" in the hospital setting.

Serratia marcescens is an opportunistic pathogen historically associated with sudden outbreaks in intensive care units (ICUs) and the spread of carbapenem-resistant genes. However, the ecology of S. marcescens populations in the hospital ecosystem remains largely unknown. We combined epidemiological information of 1,432 Serratia spp. isolates collected from sinks of a large ICU that underwent demographic and operational changes (2019-2021) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019) with 165 genomic data. These genomes were grouped into clades (1-4) and subclades (A and B) associated with distinct species: Serratia nematodiphila (1A), S. marcescens (1B), Serratia bockelmannii (2A), Serratia ureilytica (2B), S. marcescens/Serratia nevei (3), and S. nevei (4A and 4B). They may be classified into an S. marcescens complex (SMC) due to the similarity between/within subclades (average nucleotide identity >95%-98%), with clades 3 and 4 predominating in our study and publicly available databases. Chromosomal AmpC ß-lactamase with unusual basal-like expression and prodigiosin-lacking species contrasted classical features of Serratia. We found persistent and coexisting clones in sinks of subclades 4A (ST92 and ST490) and 4B (ST424), clonally related to outbreak isolates carrying blaVIM-1 or blaOXA-48 on prevalent IncL/pB77-CPsm plasmids from our hospital since 2017. The distribution of SMC populations in ICU sinks and patients reflects how Serratia species acquire, maintain, and enable plasmid evolution in both "source" (permanent, sinks) and "sink" (transient, patients) hospital patches. The results contribute to understanding how water sinks serve as reservoirs of Enterobacterales clones and plasmids that enable the persistence of carbapenemase genes in healthcare settings, potentially leading to outbreaks and/or hospital-acquired infections.IMPORTANCEThe "hospital environment," including sinks and surfaces, is increasingly recognized as a reservoir for bacterial species, clones, and plasmids of high epidemiological concern. Available studies on Serratia epidemiology have focused mainly on outbreaks of multidrug-resistant species, overlooking local longitudinal analyses necessary for understanding the dynamics of opportunistic pathogens and antibiotic-resistant genes within the hospital setting. This long-term genomic comparative analysis of Serratia isolated from the ICU environment with isolates causing nosocomial infections and/or outbreaks within the same hospital revealed the coexistence and persistence of Serratia populations in water reservoirs. Moreover, predominant sink strains may acquire highly conserved and widely distributed plasmids carrying carbapenemase genes, such as the prevalent IncL-pB77-CPsm (pOXA48), persisting in ICU sinks for years. The work highlights the relevance of ICU environmental reservoirs in the endemicity of certain opportunistic pathogens and resistance mechanisms mainly confined to hospitals.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Rapid detection of plasmid-mediated AmpC-producers by eazyplex® SuperBug AmpC assay compared to whole-genome sequencing.

Current methods for plasmid-mediated AmpC ß-lactamase (pAmpC) detection in routine microbiological laboratories are based on various phenotypic tests. Eazyplex®SuperBug AmpC assay is a molecular assay based on isothermal amplification for rapid detection of the most common pAmpC types from bacterial culture: CMY-2 group, DHA, ACC and MOX. Our aim was to evaluate the diagnostic performance of this assay. The assay was evaluated on 64 clinical isolates of Enterobacterales without chromosomal inducible AmpC, and with phenotypically confirmed AmpC production. The results were confirmed, and isolates further characterized by whole-genome sequencing (WGS). eazyplex®SuperBug AmpC assay correctly detected the two most common pAmpC types CMY-2 group (16/16) and DHA (19/19). Detection of ACC and MOX could not be evaluated on our set of isolates since there was only one isolate harbouring ACC and none with MOX. pAmpC encoding genes could be detected in only eight of 36 investigated Escherichia coli isolates. The remaining 28 E. coli isolates harboured previously described mutations in the blaEC promoter, leading to the overexpression of chromosomally encoded E. coli specific AmpC ß-lactamase. All results were 100% concordant with the results of WGS. eazyplex®SuperBug AmpC assay enabled rapid and reliable detection of pAmpC-encoding genes in Enterobacterales like Klebsiella spp. and Proteus spp. and the distinction between plasmid-mediated and chromosomally encoded AmpC in E. coli.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type B-lactamase / Pruebas rápidas de sensibilidad antibiótica de hemocultivos con enterobacterias portadoras de B-lactamasa tipo AmpC cromosómica inducible

Introduction: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. Methods: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. Results: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. Conclusions: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.(AU)

Introducción: El tratamiento precoz y adecuado de las bacteriemias disminuye la morbilidad y mortalidad de los pacientes. El objetivo es desarrollar un método preliminar de pruebas rápidas de sensibilidad antibiótica (PRSA) en enterobacterias con AmpC cromosómica inducible. Métodos: Las PRSA se realizaron directamente de hemocultivos simulados positivos para 49 enterobacterias con AmpC cromosómica inducible. Los resultados se leyeron a las 4, 6 y 8 horas de incubación. La microdilución en caldo comercial se consideró el método de referencia. Se evaluaron discos de 10 antibióticos. Resultados: La proporción de pruebas legibles a las 4 horas fue del 85%. Todas las PRSA pudieron leerse a las 6 y 8 horas. Para la mayoría de los antibióticos, el resultado S o R a las 4, 6 y 8 horas fue superior al 80%, después de que se establecieran puntos de corte provisionales y se definiera el área de incertidumbre técnica. Conclusiones: Este método preliminar parece ser de utilidad práctica, aunque debería ampliarse para ajustar los puntos de corte y diferenciar por especies.(AU)

Clonal Distribution and Its Association With the Carbapenem Resistance Mechanisms of Carbapenem-Non-Susceptible Pseudomonas aeruginosa Isolates From Korean Hospitals.

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-ß-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2 (N=3), and blaNDM-1 (N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.