Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.

Rapid culture-based LNZ test for detection of linezolid susceptibility/resistance in staphylococci and enterococci.

A rapid, easy-to-handle, cost-effective and universal culture-based test was developed for the identification of linezolid resistance among the most clinically relevant enterococcal and staphylococcal species. Our technique was tested using linezolid-resistant (n = 50) and linezolid-susceptible (n = 67) Gram-positive isolates: 34 Enterococcus faecium, 20 Enterococcus faecalis, 20 Staphylococcus aureus, 38 Staphylococcus epidermidis, and 5 Staphylococcus capitis. The susceptibility/resistance phenotype of E. faecium, E. faecalis, S. aureus, and S. epidermidis to linezolid was detected within 4.5 hours, while an extended timeframe was actually required for S. capitis (6.5 hours). The Rapid LNZ test showed a full agreement with the standard broth microdilution method, independently of the molecular resistance mechanism and MIC values, with sensitivities and specificities of 100% for all species.

Plasmid-mediated fosfomycin resistance in Escherichia coli isolates of worldwide origin.

OBJECTIVES: Fosfomycin is a first-line treatment for uncomplicated urinary tract infections (UTIs) in several European countries, and it is increasingly becoming the treatment of choice globally. Resistance to fosfomycin in Escherichia coli can be exerted through several mechanisms, including the acquisition of fosfomycin-modifying enzymes, of which the FosA-type enzymes are the most common. This study analysed, both phenotypically and genotypically, an international collection of E. coli strains harbouring acquired fosA genes. METHODS: Thirty-one fosA-positive E. coli isolates were obtained from both clinical and environmental sources, from seven countries (Portugal (n = 12), Switzerland (n = 9), China (n = 3), France (n = 2), Nepal (n = 2), South Africa (n = 2), Kuwait (n = 1)). MICs were determined according to EUCAST guidelines. Whole genome sequencing (WGS) was performed on 23 isolates, and complete fosA plasmid sequences were determined for 12. Conjugation assays were performed on seven isolates. RESULTS: All isolates exhibited high-level resistance to fosfomycin (64 to >256 mg/L). WGS of 23 isolates identified 17 sequence types (STs), and 16 harboured fosA3, four fosA4, two fosA8, and one fosA10. ESBLs, pAmpC, or carbapenemase genes were present in 15, four, and three isolates, respectively. The fosA plasmids of 12 isolates were determined and were diverse in size (∼67 kb to ∼235 kb), resistance gene carriage, and replicon types. Six fosA plasmids additionally carried ESBL or carbapenemase genes. Conjugation assays, performed on seven isolates harbouring diverse plasmids, identified that all were capable of being transmitted. CONCLUSION: This study highlights the necessity of the surveillance and close monitoring of fosfomycin resistance in E. coli, essential to maintain the optimal use of this treatment option.

Occurrence of plasmid-mediated fosfomycin resistance (fos genes) among Escherichia coli isolates, Portugal.

OBJECTIVES: To evaluate the occurrence of plasmid-mediated fos genes among fosfomycin-resistant Escherichia coli isolates collected from patients in Lisbon, Portugal, and characterize the fos-positive strains. METHODS: A total of 19 186 E. coli isolates were prospectively collected between April 2022 and January 2023 from inpatients and outpatients at a private laboratory in Lisbon. Fosfomycin resistance was initially assessed by semi-automated systems and further confirmed by the disc diffusion method. Resistant isolates were investigated for plasmid-mediated fos genes (fosA1-fosA10, fosC and fosL1-fosL2) and extended-spectrum beta-lactamases (ESBLs) by PCR and sequencing. Multilocus sequence typing was performed to evaluate the clonal relationship among fos-carrying isolates. RESULTS: Out of the 19 186 E. coli isolates, 100 were fosfomycin-resistant (0.5%), out of which 15 carried a fosA-like gene (15%). The most prevalent fosfomycin-resistant determinant was fosA3 (n = 11), followed by fosA4 (n = 4). Among the 15 FosA-producing isolates, 10 co-produced an ESBL (67%), being either of CTX-M-15 (n = 8) or CTX-M-14 (n = 2) types. The fosA3 gene was carried on IncFIIA-, IncFIB-, and IncY-type plasmids, whereas fosA4 was always located on IncFIB-type plasmids. Most FosA4-producing isolates belonged to a single sequence type ST2161, whereas isolates carrying the fosA3 gene were distributed into nine distinct genetic backgrounds. CONCLUSION: The prevalence of fosfomycin-resistant E. coli isolates is still low in Portugal. Notably, 15% of fosfomycin-resistant isolates harbour a transferable fosA gene, among which there is a high rate of ESBL producers, turning traditional empirical therapeutical options used in Portugal (fosfomycin and amoxicillin-clavulanic acid) ineffective.

ESBL- and Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae among Bivalves from Portuguese Shellfish Production Areas.

Bivalves are filter-feeding organisms and biomarkers of bacterial pollution. Our study aimed to analyze the occurrence and characteristics of extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Escherichia coli among bivalves. A total of 522 bivalve samples were collected along Portuguese shellfish production areas. Homogenized samples were screened for E. coli contamination on corresponding selective plates, allowing for concomitant growth of Klebsiella pneumoniae. E. coli growth was observed in 39% of the samples. Subsequent selective screening identified nine samples (4.4%) contaminated with ESBL producers, corresponding to E. coli (n = 7) and K. pneumoniae (n = 2), while a single carbapenemase-producing K. pneumoniae (0.5%) was identified. ESBLs were all CTX-M-types commonly identified in human isolates, i.e., CTX-M-32 (n = 4), CTX-M-15 (n = 4), and CTX-M-14 (n = 1). The carbapenemase producer harbored the blaGES-5 gene located on a ColE plasmid. Clonality was evaluated by multilocus sequence typing, identifying E. coli backgrounds as ST10, ST23, ST540, ST617, ST746, SLV206, and SLV2325, commonly identified among environmental and human strains. The K. pneumoniae isolates belonged to ST834, ST15, and DLV644. The occurrence of ESBL- and carbapenemase-producing Enterobacteriaceae in bivalves reveals how the marine environment constitutes a reservoir of critical bacterial pathogens, thus potentially representing a risk to human health.

Multiplex PCR for detection of acquired plasmid-borne fosfomycin resistance fos genes in Escherichia coli.

A rapid (<3 hours) and reliable multiplex PCR was developed for detecting simultaneously known plasmid-mediated fos genes conferring acquired resistance to fosfomycin. Our technique was tested on a collection of Escherichia coli isolates previously identified as bearing the fosA-, fosC- and fosL-like genes, showing a sensitivity and a specificity of 100%.

Urban Pigeons (Columba livia) as a Source of Broad-Spectrum ß-Lactamase-Producing Escherichia coli in Lisbon, Portugal.

Wild birds may be healthy carriers, and therefore, may be involved in the dissemination of clinically relevant antimicrobial-resistant bacteria, such as extended-spectrum ß-lactamases (ESBL) and carbapenemase-producing Enterobacteriaceae. This study evaluated whether urban pigeons living in five spots in Lisbon, Portugal, may be colonized and, therefore, constitute potential spreaders of multidrug-resistant bacteria. A total of 100 pigeon fecal samples were collected in different urban areas for the detection of ESBL- or carbapenemase-producing Enterobacteriaceae. All ß-lactamase-producing isolates were tested for antimicrobial susceptibility and their genetic backgrounds were characterized by multilocus sequence typing. Of the 100 fecal samples collected, nine ESBL-producing Escherichia coli (9%) were identified. Three isolates carried the blaCTX-M-15 gene, three isolates harbored the blaCTX-M-27 and three isolates carried the blaSHV-12 gene. Genotyping of the nine ESBL-producing E. coli strains revealed seven different sequence types (STs) including ST10, ST131, ST154, ST206, ST1488 (SLV ST10), ST2858 and ST3576, most of which have been already described in humans, animals or in the environment. Urban pigeons constitute a potential source of ESBL genes and may be a transmission vehicle of multidrug-resistant bacteria in the environment.

Screening and Characterization of Multidrug-Resistant Enterobacterales among Hospitalized Patients in the African Archipelago of Cape Verde.

This study aimed to investigate, for the first time, the occurrence and characteristics of extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacterales in Cape Verde. A total of 98 inpatients hospitalized at Hospital Universitário Agostinho Neto were screened for rectal colonization. All ESBL- and carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by multilocus sequence typing. Mating-out assay followed by PCR-based replicon typing were performed to characterize the plasmids harboring carbapenemase encoding genes. A large proportion of patients carried ESBL- or carbapenemase-producing Enterobacterales (56% and 6%, respectively). Among 93 ESBL-producing isolates, there were mainly Klebsiella pneumoniae (58%) and Escherichia coli (37%). Five different ESBLs were detected, with CTX-M-15 being highly predominant (92%). Six carbapenemase-producing isolates (five E. coli and one K. pneumoniae) were recovered, and all of the OXA-48-like type (four OXA-181, one OXA-48, and one OXA-244). The blaOXA-48 gene was located on an IncFI-type plasmid, the blaOXA-181 gene on IncFI or IncX3 plasmids, and the blaOXA-244 gene was found to be chromosomally located. The five carbapenemase-producing E. coli isolates belonged to five distinct sequence types. This study overall showed a very high prevalence of ESBL-producing Enterobacterales, as well as the emergence of carbapenemase producers in this hospital.

Whole-genome analysis uncovers loss of blaZ associated with carriage isolates belonging to methicillin-resistant Staphylococcus aureus (MRSA) clone ST5-VI in Cape Verde.

OBJECTIVES: Surveillance studies for Staphylococcus aureus carriage are a primary tool to survey the prevalence of methicillin-resistant S. aureus (MRSA) in the general population, patients and healthcare workers. We have previously reported S. aureus carriage in various African countries, including Cape Verde. METHODS: Whole-genome sequences of 106 S. aureus isolates from Cape Verde were determined. RESULTS: Staphylococcus aureus carriage isolates in Cape Verde show high genetic variability, with the detection of 27 sequence types (STs) and three primary genetic clusters associated with ST152, ST15 and ST5. One transmission event with less than eight core-genome single nucleotide polymorphisms (cgSNP) differences was detected among the ST5-VI MRSA lineage. Genetic analysis confirmed the phenotypic resistance and allowed the identification of six independent events of plasmid or transposon loss associated with the deletion of blaZ in nine isolates. In the four ST5 MRSA isolates, loss of the blaZ plasmid coincided with the acquisition of SCCmec type VI and an unusual penicillin phenotype with a minimum inhibitory concentration (MIC) at the breakpoint, indicating an adaptation trend in this endemic lineage. Similar events of blaZ plasmid loss, with concomitant acquisition SCCmec elements, were detected among ST5 isolates from different geographical origins. CONCLUSION: Overall, the genome data allowed to place isolates in a phylogenetic context and to identify different blaZ gene deletions associated with plasmid or transposon loss. Genomic analysis unveiled adaptation and evolution trends, namely among emerging MRSA lineages in the country, which deserve additional consideration in the design of future infection control protocols.

Prevalence of biocide resistance genes and chlorhexidine and mupirocin non-susceptibility in Portuguese hospitals during a 31-year period (1985-2016).

OBJECTIVES: Methicillin-resistantStaphylococcus aureus (MRSA) remains a major human pathogen. MRSA decolonisation strategies frequently combine chlorhexidine baths and mupirocin nasal ointment. Although MRSA remains widespread in Portuguese hospitals, information regarding resistance to biocides and mupirocin is scarce. We evaluated the prevalence of biocide resistance genes and chlorhexidine and mupirocin non-susceptibility in a representative and well-characterised collection of MRSA isolated in Portuguese hospitals during a 31-year period (1985-2016). METHODS: Prevalence of five biocide resistance genes (lmrS, mepA, sepA, qacAB and smr) was determined by PCR. Antibiotic susceptibility was assessed by disk diffusion and by MIC determination using broth microdilution (chlorhexidine) and Etest (mupirocin). RESULTS: Chromosomal genessepA and mepA were detected in all isolates, while lmrS was found in 87.1%. The prevalence of plasmid-borne genes was significant for qacAB (22.4%), associated with the Iberian (ST247-I/IA) clone (P < 0.0001), and low for smr (1.0%) detected among isolates belonging to the ST239-III/IIIvariant clone. Chlorhexidine non-susceptibility (MIC ≥ 4 mg/L) was observed in two isolates belonging to the EMRSA-15 clone (ST22-IV). Non-susceptibility to mupirocin (MIC > 1 mg/L) was significant (15.4%; n = 31) and mainly found among isolates of the EMRSA-15 clone (P < 0.0001; n = 29). One isolate presented low-level mupirocin resistance (MIC = 32 mg/L), and two missense mutations N213D (A637G) and V588F (G1762T) were identified in the ileS gene. CONCLUSION: Concerningly, we detected a high prevalence of biocide resistance genes and an association of mupirocin and chlorhexidine non-susceptibility with the dominant EMRSA-15 clone in Portuguese hospitals.

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.

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.

Epidemiology of carbapenemase-producing Klebsiella pneumoniae in northern Portugal: Predominance of KPC-2 and OXA-48.

OBJECTIVES: To provide, for the first time, data on the molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae clinical isolates from the northern region of Portugal (Trás-os-Montes and Alto Douro). METHODS: A total of 106 carbapenemase-producing K. pneumoniae isolates recovered from clinical samples and rectal swabs between January 2018 and March 2019 were included in this study. All isolates were characterized by antimicrobial susceptibility, identification of resistance determinants, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid analysis. RESULTS: The most common carbapenemase identified was KPC-2 (91%), followed by OXA-48 (9%). The blaKPC-2 gene was carried onto IncN (60%) and IncF (40%) plasmid types, whereas the blaOXA-48 gene was mainly located on the IncL (90%) incompatibility group. Molecular characterization distributed the 106 isolates into 29 PFGE types and 21 sequence types (STs), but three clones included 50% of the isolates: PFGE A-ST147-KPC-2 (29%), B-ST15-KPC-2 (15%), and C-ST11-OXA-48 (6%). Antimicrobial resistance rates were the following: ciprofloxacin (76%), trimethoprim-sulfamethoxazole (75%), tobramycin (62%), gentamicin (34%), amikacin (25%), tigecycline (21%), fosfomycin (10%), and colistin (7%). None of the colistin-resistant isolates harboured mcr genes. All isolates remained susceptible to ceftazidime/avibactam, but 10% presented elevated MICs (3 and 4mg/L). CONCLUSIONS: KPC-2 was the predominant carbapenemase among K. pneumoniae isolates currently circulating at this hospital from northern Portugal, followed by OXA-48. These data contrast with those obtained from the rest of the country, where KPC-3 predominates. This study showed a polyclonal structure of KPC-2-producing K. pneumoniae isolates with a predominance of the ST147 and ST15 clones.

A five-year retrospective study shows increasing rates of antimicrobial drug resistance in Cabo Verde for both Staphylococcus aureus and Escherichia coli.

OBJECTIVES: Data on baseline drug resistance important in informing future antimicrobial stewardship programs. So far, no data on the antimicrobial drug resistance of clinical isolates available for the African archipelago of Cabo Verde. METHODS: We performed a retrospective analysis over years (2013-17) of the drug susceptibility profiles of clinical isolates in the two main hospitals of Cabo Verde. For Escherichia coli and Staphylococcus aureus, representing 47% and 26% of all clinical isolates, the antimicrobial drug resistance profile was reported for six representative drugs. RESULTS: For E. coli we detected an increase in resistance to ampicillin, amoxicillin/clavulanic acid, ceftriaxone, ciprofloxacin and trimethoprim-and for S. aureus to methicillin, erythromycin and trimethoprim-sulfamethoxazole. This increase in both the most commonly isolated bacterial pathogens is alarm as it might compromise empirical treatment in a setting with limited access to laboratory testing. CONCLUSIONS: When compared to the published low resistance rates in carriage isolates, the more alarming situation in clinical isolates for S. aureus might encourage antimicrobial stewardship programs to reduce in hospital settings, possibly as part of the Cabo Verdean national plan against antimicrobial drug resistance.

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation.

OBJECTIVES: To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. METHODS: Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. RESULTS: We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC ß-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum ß-lactamase possessing weak carbapenemase activity, encoded by another plasmid. CONCLUSIONS: We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

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.

Intestinal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae at admission in a Portuguese hospital.

To evaluate the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae fecal carriers at admission in a Portuguese hospital and to determine the epidemiology and antimicrobial resistance patterns of ESBL-producing isolates. During a 2-month period, rectal swabs were collected at hospital admission from 151 at-risk patients. In addition, 48 rectal swabs were obtained from weekly screenings of 37 patients hospitalized for > 48 h. All ESBL/carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by PFGE and MLST. The prevalence of ESBL producers at hospital admission was 17% and 24% among at-risk patients hospitalized for > 48 h, while the prevalence of carbapenemase producers was 3% in both cases. Most of the isolates were Escherichia coli (54%) and Klebsiella pneumoniae (41%). The most common ESBL identified was CTX-M-15 (n = 17/34; 50%), followed by CTX-M-27 (n = 10; 29%), CTX-M-33 (n = 4; 12%), SHV-12 (n = 2), and CTX-M-55 (n = 1). The 20 E. coli isolates were distributed into 16 PFGE types and nine sequence types (ST), with 60% of the isolates belonging to ST131. The 15 K. pneumoniae were grouped into 12 PFGE types and nine STs, with three STs (ST17, ST449, ST147) corresponding to 60% of the isolates. A high proportion of isolates showed resistance to ciprofloxacin (86%), trimethoprim-sulfamethoxazole (68%), tobramycin (57%), and gentamicin (43%). All isolates remained susceptible to fosfomycin. A high prevalence of ESBL-producing Enterobacteriaceae was found at hospital admission among at-risk patients and > 50% of the isolates showed resistance to first-line antibiotics for the treatment of lower urinary tract infections, leaving fosfomycin as an alternative.

Epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus isolates colonizing pigs with different exposure to antibiotics.

BACKGROUND: In 2016, very high rates of methicillin-resistant Staphylococcus aureus (MRSA)-ST398 (99%) were found in Portuguese pig farms that used colistin, amoxicillin, and zinc oxide as feed additives. Since then, farms A and B banned the use of colistin, and farm C banned the use of both antibiotics. OBJECTIVE: The aim of the present study was to evaluate the impact of the ban of colistin and amoxicillin on pig MRSA carriage rates, clonal types and antimicrobial resistance, compared to the results obtained in 2016. METHODS: In 2018, 103 pigs (52 from farm B using amoxicillin only as a feed additive and 51 from farm C where no antibiotics were included in the feed regimen) were nasally swabbed for MRSA colonization. Isolates were tested for antimicrobial susceptibility, and characterised by spa typing, SCCmec typing and MLST. Whole genome sequencing (WGS) was performed for representative isolates. RESULTS: Overall, 96% of the pigs swabbed in 2018 carried MRSA, mostly ST398-SCCmec V-spa types t011/t108. MRSA from pigs not receiving antibiotics in the feed regimen showed susceptibility to a higher number of antibiotics, namely erythromycin, ciprofloxacin, gentamicin, and chloramphenicol. Notably, most of these isolates (n = 52) presented an unusual erythromycin-susceptibility/clindamycin-resistance phenotype. WGS showed that these isolates lacked the erm and the lnu genes encoding resistance to macrolides and lincosamides, respectively, but carried the vgaALC gene encoding resistance to lincosamides, which is here firstly identified in S. aureus ST398. CONCLUSION: After two years the ban of colistin and amoxicillin as feed additives had no significant impact on the MRSA nasal carriage rates. Nevertheless, the MRSA strains circulating in those farms showed resistance to a lower number of antibiotic classes.

CTX-M-33, a CTX-M-15 derivative conferring reduced susceptibility to carbapenems.

CTX-M-type extended-spectrum ß-lactamases (ESBL) are widespread among Enterobacterales worldwide. The most common variant is CTX-M-15 hydrolyzing ceftazidime at high rate, but sparing carbapenems. We identified here CTX-M-33, a point mutant derivative of CTX-M-15 (Asp to Ser substitution at Ambler position 109), exhibiting a low carbapenemase activity. ß-Lactamase CTX-M-33 was identified in a Klebsiella pneumoniae isolate belonging to ST405, lacking the outer membrane protein OmpK36, that was resistant to broad-spectrum cephalosporins and ß-lactam/ß-lactamase inhibitor combinations, and displayed a decreased susceptibility to carbapenems. Comparative hydrolytic activity assays showed that CTX-M-33 hydrolyzed ceftazidime at a lower level than CTX-M-15, but significantly hydrolyzed meropenem. In addition, CTX-M-33 showed higher Mutant Prevention Concentration values and wider mutant selection window in presence of meropenem, in accordance with its observed hydrolytic properties. We identified here the very first CTX-M enzyme possessing a weak carbapenemase activity, that may correspond to an emerging phenomenon when considering its possibility to evolve from the widespread ESBL CTX-M-15.

Epidemiology of Carbapenemase-Producing Klebsiella pneumoniae in a Hospital, Portugal.

We aimed to provide updated epidemiologic data on carbapenem-resistant Klebsiella pneumoniae in Portugal by characterizing all isolates (N = 46) recovered during 2013-2018 in a 123-bed hospital in Lisbon. We identified blaKPC-3 (n = 36), blaOXA-181 (n = 9), and blaGES-5 (n = 8) carbapenemase genes and observed co-occurrence of blaKPC-3 and blaGES-5 in 7 isolates. A single GES-5-producing isolate co-produced the extended-spectrum ß-lactamase BEL-1; both corresponding genes were co-located on the same ColE1-like plasmid. The blaOXA-181 gene was always located on an IncX3 plasmid, whereas blaKPC-3 was carried on IncN, IncFII, IncFIB, and IncFIIA plasmid types. The 46 isolates were distributed into 13 pulsotypes and 9 sequence types. All isolates remained susceptible to ceftazidime/avibactam, but some exhibited reduced antimicrobial susceptibility (MIC = 3 mg/L).