Inter-phylum circulation of a beta-lactamase-encoding gene: a rare but observable event.

Beta-lactamase-mediated degradation of beta-lactams is the most common mechanism of beta-lactam resistance in Gram-negative bacteria. Beta-lactamase-encoding genes can be transferred between closely related bacteria, but spontaneous inter-phylum transfers (between distantly related bacteria) have never been reported. Here, we describe an extended-spectrum beta-lactamase (ESBL)-encoding gene (blaMUN-1) shared between the Pseudomonadota and Bacteroidota phyla. An Escherichia coli strain was isolated from a patient in Münster (Germany). Its genome was sequenced. The ESBL-encoding gene (named blaMUN-1) was cloned, and the corresponding enzyme was characterized. The distribution of the gene among bacteria was investigated using the RefSeq Genomes database. The frequency and relative abundance of its closest homolog in the global microbial gene catalog (GMGC) were analyzed. The E. coli strain exhibited two distinct morphotypes. Each morphotype possessed two chromosomal copies of the blaMUN-1 gene, with one morphotype having two additional copies located on a phage-plasmid p0111. Each copy was located within a 7.6-kb genomic island associated with mobility. blaMUN-1 encoded for an extended-spectrum Ambler subclass A2 beta-lactamase with 43.0% amino acid identity to TLA-1. blaMUN-1 was found in species among the Bacteroidales order and in Sutterella wadsworthensis (Pseudomonadota). Its closest homolog in GMGC was detected frequently in human fecal samples. This is, to our knowledge, the first reported instance of inter-phylum transfer of an ESBL-encoding gene, between the Bacteroidota and Pseudomonadota phyla. Although the gene was frequently detected in the human gut, inter-phylum transfer was rare, indicating that inter-phylum barriers are effective in impeding the spread of ESBL-encoding genes, but not entirely impenetrable.

Selective Culture Medium for Screening of Fosfomycin Resistance in Enterobacterales.

A selective medium for screening fosfomycin (FOS)-resistant Enterobacterales was developed. Performances of this medium were first evaluated by using cultures of a collection of 84 enterobacterial clinical strains (42 FOS susceptible and 42 FOS resistant). The SuperFOS medium showed excellent sensitivity and specificity of detection (100%) in those conditions. Then, by testing spiked stool and spiked urine specimens, it revealed excellent performances, with lower limits of identification ranging from 101 to 102 CFU/ml. This screening medium allows easy and accurate detection of FOS-resistant isolates regardless of their resistance mechanisms.

KSA-1, a naturally occurring Ambler class A extended spectrum ß-lactamase from the enterobacterial species Kosakonia sacchari.

BACKGROUND: Several bacterial species belonging to the Gammaproteobacteria possess intrinsic class A ß-lactamase genes that may represent a source of further dissemination and acquisition to other Gram-negative species. Here we characterised KSA-1 class A ß-lactamase, the gene of which was identified within the chromosome of an environmental Enterobacterales species, namely Kosakonia sacchari, which was also recently identified as the progenitor of an MCR-like colistin-resistance determinant. METHODS: In silico analysis using the GenBank database identified a class A ß-lactamase gene within the chromosome of K. sacchari SP1 (GenBank accession no. WP_017456759). The corresponding protein KSA-1 shared 63% amino acid identity with the intrinsic CKO-1 from Citrobacter koseri and 53% with TEM-1. Using the K. sacchari DSM 100203 reference strain as a template, blaKSA-1 was amplified, cloned into the plasmid pUCp24 and expressed in Escherchia coli TOP10. Minimal inhibitory concentrations and kinetic parameters were obtained from the purified enzyme. RESULTS: K. sacchari strain SP1 conferred resistance to amino-, carboxy- and ureido-penicillins only. Once produced within E. coli, KSA-1 showed a typical clavulanic acid-inhibited extended spectrum ß-lactamase associated with a peculiar temocillin resistance profile. Kinetic assays were performed using a purified extract of KSA-1 and demonstrated a high hydrolysis rate for benzylpenicillin and piperacillin, as well as weakly extended spectrum cephalosporins. Determination of inhibitory constants showed 50% inhibitory concentration values of 2.2, 3 and 1.8 nM for clavulanic acid, tazobactam and avibactam, respectively. Analysis of sequences surrounding the blaKSA-1 gene did not reveal any mobile element that could have been involved in the acquisition of this ß-lactamase gene in that species. CONCLUSION: KSA-1 is a class A extended spectrum ß-lactamase distantly related to known extended spectrum or broad-spectrum Ambler class A ß-lactamases, which is highly resistant to temocillin. The blaKSA-1 gene could be considered as intrinsic within the species.

Increasing Trends of Association of 16S rRNA Methylases and Carbapenemases in Enterobacterales Clinical Isolates from Switzerland, 2017-2020.

Aminoglycosides (AGs) in combination with ß-lactams play an important role in antimicrobial therapy in severe infections. Pan-resistance to clinically relevant AGs increasingly arises from the production of 16S rRNA methylases (RMTases) that are mostly encoded by plasmids in Gram-negative bacteria. The recent emergence and spread of isolates encoding RMTases is worrisome, considering that they often co-produce extended-spectrum ß-lactamases (ESBLs) or carbapenemases. Our study aimed to retrospectively analyze and characterize the association of carbapenem- and aminoglycoside-resistant clinical isolates in Switzerland during a 3.5-year period between January 2017 and June 2020. A total of 103 pan-aminoglycoside- and carbapenem-resistant clinical isolates were recovered at the NARA (Swiss National Reference Center for Emerging Antibiotic Resistance) during the 2017-2020 period. Carbapenemase and RMTase determinants were identified by PCR and sequencing. The characterization of plasmids bearing resistance determinants was performed by a mating-out assay followed by PCR-based replicon typing (PBRT). Clonality of the isolates was investigated by multilocus sequence typing (MLST). Over the 991 Enterobacterales collected at the NARA during this period, 103 (10.4%) of them were resistant to both carbapenems and all aminoglycosides. Among these 103 isolates, 35 isolates produced NDM-like carbapenemases, followed by OXA-48-like (n = 23), KPC-like (n = 21), or no carbapenemase (n = 13), OXA-48-like and NDM-like co-production (n = 7), and VIM-like enzymes (n = 4). The RMTases ArmA, RmtB, RmtC, RmtF, RmtG, and RmtB + RmtF were identified among 51.4%, 13.6%, 4.9%, 24.3%, 1%, and 1%, respectively. Plasmid co-localization of the carbapenemase and the RMTase encoding genes was found among ca. 20% of the isolates. A high diversity was identified in terms of the nature of associations between RMTase and carbapenemase-encoding genes, of incompatibility groups of the corresponding plasmids, and of strain genetic backgrounds, highlighting heterogeneous importations rather than clonal dissemination.

A Selective Culture Medium for Screening Carbapenem Resistance in Pseudomonas spp.

Purpose: The SuperCP medium containing meropenem (2 mg/L) was evaluated for screening of carbapenem-resistant Pseudomonas species. Materials and Methods: It was evaluated using 29 meropenem-susceptible and 56 meropenem-nonsusceptible Pseudomonas-like clinical isolates, the latter exhibiting a variety of carbapenem-resistance mechanisms. Results: Its sensitivity and specificity of detection were found to be 91% and 100%, respectively. By testing spiked stools, an excellent performance of the medium was also observed for detection of carbapenem-resistant Pseudomonas aeruginosa, with a lowest detection limit ranging from 100 to 102 CFU/mL. Conclusion: This screening medium provides the opportunity to select carbapenem-resistant Pseudomonas and Pseudomonas-related isolates regardless of their resistance mechanism.

Does an Antibiotic Stewardship Applied in a Pig Farm Lead to Low ESBL Prevalence?

Background. The aim of the present study was to prospectively evaluate the prevalence of intestinal carriage of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales among pigs from a Swiss farm attending an animal health and antibiotic stewardship program and to determine the associated mechanisms of resistance. Materials/Methods. Eighty-one fecal samples were recovered and screened for either ß-lactam-resistant, colistin-resistant, or aminoglycoside-resistant Enterobacterales, using respective screening media. All recovered isolates were tested for antimicrobial susceptibility and their clonal relationship (PFGE and MLST). Plasmid typing was performed by plasmid-based replicon typing (PBRT). Resistance genes were searched by PCR and sequencing. Results. A total of 38 ESBL-producing Escherichia coli and a single ESBL-producing Enterobacter cloacae were recovered from 81 pigs, corresponding to a prevalence of 50%, no other ß-lactamase producer being identified. Among the 38 ESBL-producing E. coli, all belonged to sequence type (ST) ST10, except two ST34 and ST744 isolates. Among the ST10-blaCTX-M-1 isolates, three subclones (n = 22, n = 13, and n = 1, respectively) were identified according to the PFGE analysis. The most commonly identified IncI1 plasmid harboring the blaCTX-M-1 gene was 143 kb in size and coharbored other resistance genes. Only three colistin-resistant Enterobacterales isolates were recovered, namely two Klebsiella pneumoniae isolates and a single E. cloacae isolate. Screening for the plasmid-borne mcr-1 to mcr-9 genes in these three isolates gave negative results. The two K. pneumoniae isolates were clonally related, belonged to ST76, and harbored a truncated mgrB chromosomal gene being the source of colistin resistance. Conclusion. A high prevalence of fecal carriage of ESBL-producing E. coli was found, being mainly caused by the spread of a clonal lineage within the farm. By contrast, a low prevalence of colistin-resistant Enterobacterales was found.

Implementation and evaluation of methods for the optimal detection of carbapenem-resistant and colistin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii from stools.

Carbapenem-resistant Acinetobacter baumannii (CRAB) and Pseudomonas aeruginosa (CRPA), as well as polymyxin-resistant A. baumannii (PMR-AB) and P. aeruginosa (PMR-PA), were used to test different enrichment strategies from spiked stools. Three procedures were compared, namely, direct inoculation on selective plates and plating after a 24-h enrichment step in tryptic soy broth with or without antibiotics. Selective agar plates were used including CHROMagar-Pseudomonas supplemented with meropenem (2 mg/L), and CHROMagar-MDR-Acinetobacter agar and CHROMagar COL-APSE plates. Use of enrichment broths significantly enhanced the recovery of CRAB, CRPA, PMR-AB, and PMR-PA. However, supplementing or not the pre-enrichment broth with antibiotics had no impact. The proposed strategy for screening multidrug-resistant nonfermenters is of low cost, is easy to implement, and might be useful for outbreak containment.

Occurrence of CTX-M-15- and MCR-1-producing Enterobacterales in pigs in Portugal: Evidence of direct links with antibiotic selective pressure.

AIMS: To undertake a prospective analysis of the occurrence of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales colonizing pigs at two farms in Portugal, and to evaluate the putative correlations with usage of different antibiotics. MATERIALS AND METHODS: One hundred and two faecal samples recovered from two different Portuguese pig farms were screened for polymyxin-resistant and ESBL-positive Enterobacterales. The authors had undertaken a study at one of the farms previously, but the use of colistin has since been banned; zinc oxide and amoxicillin are used as prophylactic and curative drugs, respectively, at this farm. The other farm included in this study used zinc oxide alone. RESULTS: Ninety-three ESBL-producing isolates (62 Escherichia coli, 29 Klebsiella pneumoniae, one Enterobacter aerogenes and one Enterobacter cloacae) and 17 colistin-resistant isolates (12 E. coli, four K. pneumoniae and one E. cloacae) were recovered. Among the ESBL producers, the majority (84%) produced CTX-M-15, while the others produced CTX-M-1 or CTX-M-9. Many different strain and plasmid backgrounds were identified, ruling out a massive dissemination of one major clone. In total, 17 colistin-resistant isolates were recovered, all from the first farm. All produced MCR-1, corresponding to 12 E. coli (10 clones) and three K. pneumoniae (two clones). The MCR-1 producers were all recovered from the farm where colistin had been used 2 years previously. CONCLUSION: This study showed a surprisingly high rate of CTX-M-15 producers at two Portuguese pig farms. A link was found between antibiotic selective pressure (ß-lactam or polymyxin) and the corresponding resistance rate.

High Colonization Rate and Heterogeneity of ESBL- and Carbapenemase-Producing Enterobacteriaceae Isolated from Gull Feces in Lisbon, Portugal.

In order to evaluate whether seagulls living on the Lisbon coastline, Portugal, might be colonized and consequently represent potential spreaders of multidrug-resistant bacteria, a total of 88 gull fecal samples were screened for detection of extended-spectrum ß-lactamase (ESBL)- or carbapenemase-producing Enterobacteriaceae for methicillin-resistant Staphylococcus aureus (MRSA) and for vancomycin-resistant Enterococci (VRE). A large proportion of samples yielded carbapenemase- or ESBL-producing Enterobacteriaceae (16% and 55%, respectively), while only two MRSA and two VRE were detected. Mating-out assays followed by PCR and whole-plasmid sequencing allowed to identify carbapenemase and ESBL encoding genes. Among 24 carbapenemase-producing isolates, there were mainly Klebsiella pneumoniae (50%) and Escherichia coli (33%). OXA-181 was the most common carbapenemase identified (54%), followed by OXA-48 (25%) and KPC-2 (17%). Ten different ESBLs were found among 62 ESBL-producing isolates, mainly being CTX-M-type enzymes (87%). Co-occurrence in single samples of multiple ESBL- and carbapenemase producers belonging to different bacterial species was observed in some cases. Seagulls constitute an important source for spreading multidrug-resistant bacteria in the environment and their gut microbiota a formidable microenvironment for transfer of resistance genes within bacterial species.

S-methyl Methanethiosulfonate: Promising Late Blight Inhibitor or Broad Range Toxin?

(1) Background: S-methyl methanethiosulfonate (MMTS), a sulfur containing volatile organic compound produced by plants and bacterial species, has recently been described to be an efficient anti-oomycete agent with promising perspectives for the control of the devastating potato late blight disease caused by Phytophthora infestans. However, earlier work raised questions regarding the putative toxicity of this compound. To assess the suitability of MMTS for late blight control in the field, the present study thus aimed at evaluating the effect of MMTS on a wide range of non-target organisms in comparison to P. infestans. (2) Methods: To this end, we exposed P. infestans, as well as different pathogenic and non-pathogenic fungi, bacteria, the nematode Caenorhabditis elegans as well as the plant Arabidopsis thaliana to MMTS treatment and evaluated their response by means of in vitro assays. (3) Results: Our results showed that fungi (both mycelium and spores) tolerated MMTS better than the oomycete P. infestans, but that the compound nevertheless exhibited non-negligible toxic effects on bacteria, nematodes and plants. (4) Conclusions: We discuss the mode of action of MMTS and conclude that even though this compound might be too toxic for chemical application in the field, its strong anti-oomycete activity could still be exploited when naturally released at the site of infection by plant-associated microbes inoculated as biocontrol agents.

Epidemiology of extended-spectrum ß-lactamase-producing Enterobacteriaceae among healthcare students, at the Portuguese Red Cross Health School of Lisbon, Portugal.

OBJECTIVE: The aim of the present study was to prospectively evaluate the prevalence of intestinal carriage by extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae among Portuguese students attending a Bachelors' course in healthcare, and to determine the molecular features of ESBL-producing isolates. METHODS: One-hundred and eleven faecal samples recovered from Portuguese healthcare students were screened for either ESBL-producing, carbapenem-resistant, colistin-resistant or pan-aminoglycoside-resistant Enterobacteriaceae, using respective screening media. All recovered isolates were tested for antimicrobial susceptibility and characterised by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: A total of 17 ESBL-producing Enterobacteriaceae (16 Escherichia coli and a single Klebsiella pneumoniae) were recovered from 16 students, representing a prevalence of 14.5%. The E. coli isolates were distributed into three sequence types (STs) and seven PFGE types. The most common ESBL identified was CTX-M-1 (n=13; 76%), followed by CTX-M-15 (n=3; 18%) and CTX-M-8 (n=1; 6%). The majority of the strains were resistant to sulfonamides (88%) and fosfomycin (71%). Resistance to aminoglycosides was observed at a low rate, that is 12% for both tobramycin and kanamycin. No colistin-, carbapenem- or pan-aminoglycoside-resistant isolates were recovered. A major clone, ST10-blaCTX-M-1, included 12 E. coli isolates. The blaCTX-M-1 gene was always located on an IncFIA/FIB plasmid type, co-harbouring genes encoding resistance to tetracycline, sulfonamides, trimethoprim-sulfamethoxazole and fosfomycin. CONCLUSION: The most commonly identified ESBL gene in E. coli was blaCTX-M-1, usually identified among ESBL-producing isolates recovered from animals. A high prevalence of faecal carriage of ESBL-producing E. coli was found among healthy healthcare students, underlying this population as an important reservoir.

Characterization of PAN-1, a Carbapenem-Hydrolyzing Class B ß-Lactamase From the Environmental Gram-Negative Pseudobacteriovorax antillogorgiicola.

The gene encoding the metallo-ß-lactamase (MßL) PAN-1 was identified in the genome of the environmental Gram-negative species Pseudobacteriovorax antillogorgiicola. PAN-1 shares 57% amino-acid identity with the acquired MßL SPM-1, its closest relative. Kinetic parameters performed on purified PAN-1 showed it displayed a hydrolytic activity toward most ß-lactams including carbapenems but spared cefepime and aztreonam. These results further highlight that environmental bacterial species may be reservoirs of MßL encoding genes.