Comparison of two MALDI-TOF MS systems for the identification of clinically relevant anaerobic bacteria in Argentina.

The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.

Characterization of a Clostridioides difficile ST-293 isolate from a recurrent infection in Argentina.

Clostridioides difficile is an opportunistic spore-forming pathogen responsible for antibiotic-associated diarrhea in humans. C. difficile produces two main toxins: TcdA and TcdB as well as a third toxin named binary toxin (CDT) that is also involved in virulence. The present study aimed at characterizing the C. difficile isolate ALCD3 involved in a relapse episode of nosocomial infection. Molecular characterization showed that isolate ALCD3 belongs to toxinotype 0/v and the MLST analysis demonstrated allelic profile adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 and tpi:1 which corresponds to ST293 (MLST clade: 1). During growth, isolate ALCD3 showed an early increase in the sporulation ratio as well as maximal values of heat resistant forms after 2 days of incubation. Both sporulation kinetics and production of heat resistant forms were faster for isolate ALCD3 than for the reference strain VPI 10463. Germination in the presence of the natural germinant taurocholate was faster for isolate ALCD3 than for strain VPI 10463, which indicates that isolate ALCD3 starts cortex hydrolysis earlier than strain VPI 10463. Furthermore, the co-germinant glycine, induces rapid release of dipicolinic acid (DPA) in isolate ALCD3. These findings indicate that isolate ALCD3 is particularly efficient in both sporulation and germination. The present work represents the first report of the circulation of C. difficile ST293 in Argentina. The ability of isolate ALCD3 to produce toxins and its high sporulation/germination capacity are key features compatible with a microorganism with high dissemination potential and the possibility of inducing recurrent infections.

The dilemma of identifying Peptoniphilus species by using two MALDI-TOF MS systems.

Two commercial MALDI-TOF MS systems were used to identify 18 isolates, belonging to the Peptoniphilus genus; also the 16S rRNA sequencing identity was compared against the MALDI-TOF MS system results. Bruker Biotyper system provided higher accuracy than Vitek MS system, however, adding spectra could allow a more reliable species level identification.

Emergence and clonal expansion of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1.

MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describes the emergence and clonal dissemination of K. pneumoniae ST307, recovered during November 2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3 and NDM-1. These isolates were resistant to all ß-lactams and to the ceftazidime/avibactam combination. Molecular studies showed that blaKPC-3 was located in Tn4401a platform, while blaNDM-1 was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 and downstream by bleMBL-trpF, located in a 145.5kb conjugative plasmid belonging to the Inc A/C group. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 could lead to a worrisome scenario due to the remarkable features of this clone and its resistance profile.

Full characterization of an IncR plasmid harboring qnrS1 recovered from a VIM-11-producing Pseudomonas aeruginosa.

Metallo-ß-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaVIM-11 in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening for MBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrS1; however, both markers were located in different plasmids. The qnrS1-harboring plasmid (pP6qnrS1) was 117945bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrS1, it harbored several aminoglycoside resistance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrS1, plus the first detection of an IncR plasmid in Argentina.

Spread of Clonally Related Escherichia coli Strains Harboring an IncA/C1 Plasmid Encoding IMP-8 and Its Recruitment into an Unrelated MCR-1-Containing Isolate.

Ten IMP-8-producing Escherichia coli isolates were recovered from surveillance cultures of a neonatal intensive care unit; eight of the isolates were clonally related. A 168.2-kb blaIMP-8 plasmid was fully sequenced, and it corresponded to the recently described IncA/C1-ST13 plasmid. This plasmid was detected in all isolates, even in those that were not clonally related. One unrelated isolate was also resistant to colistin and positive for mcr-1 This marker was located in a 62.7-kb IncI2 plasmid, which was also fully sequenced.

Detection and molecular characterization of Clostridium difficile ST 1 in Buenos Aires, Argentina.

Thirty one C. difficile isolates recovered in 2015 were characterized. Nineteen/31 were positive for tcdA/B, among them, 4 isolates were also positive for CDT coding genes. Two/4 cdtA/B positives isolates corresponded to ST 1 resembling BI/NAP1/027/ST 1 strain, while the others corresponded to ST 226 and ST 377.

Identification of CfiA coding genes in Bacteroides fragilis isolates recovered in Argentina. Inconsistencies in CfiA organization and nomenclature.

CfiA (CcrA) metallo-ß-lactamase is the main carbapenem resistance mechanism in B. fragilis. From cfiA positive isolates detected in a previous surveillance study, 3 displayed resistance to imipenem while the remaining were susceptible. The aim of this study was to identify the cfiA alleles and to analyze the presence of IS elements in their upstream regions. CfiA-1, CfiA-4, CfiA-13, CfiA-19 and CfiA-22 were detected. IS elements belonging to IS21 family and IS942 group were identified upstream to cfiA in the 3 imipenem resistant isolates. We present an exhaustive analysis of cfiA/CfiA registers in databases, illustrating the inconsistencies in both organization and nomenclature. According to this analysis CfiA family comprises nowadays 15 different CfiA variants coded by 24 cfiA sequences. Curation of CfiA database is mandatory, if not new cfiA admission at GenBank will contribute to make this classification more complex.

Detection and genetic characterization of ß-lactamases in Prevotella intermedia and Prevotella nigrescens isolated from oral cavity infections and peritonsillar abscesses.

A prospective analysis on ß-lactam resistance mechanisms and ß-lactamase prevalence was conducted on Prevotella intermedia and Prevotella nigrescens recovered from patients with chronic periodontitis and peritonsillar abscesses. Both phenotypic and genotypic methods were performed to characterize the ß-lactamases, their coding genes and their genetic contexts. Overall, ß-lactamase production was observed in 64% (16/25) P. intermedia and 23.8% (5/21) P. nigrescens (p < 0.01). Besides higher ß-lactamase production rates were observed in P. intermedia (8/16) than in P. nigrescens (2/16) recovered from chronic periodontitis, almost all isolates from peritonsillar abscesses were producers (8/9 and 3/3, respectively). cfxA, but not cepA and cblA, was detected in those isolates, which were previously categorized as ß-lactamase producers. CfxA producing isolates displayed higher ß-lactam MICs than non-producers in both species. The most frequent allele was cfxA2, followed by cfxA3 and a new allelic variant named cfxA6. The analysis of the downstream flanking region in the three cfxA variants revealed the association with mobA of Tn4555, suggesting their localization in a mobilizable element. ß-lactam resistance and cfxA carriage prevalence seems to be not only related to the bacterial species but also to the infection site.

First isolate of KPC-2-producing Klebsiella pneumonaie sequence type 23 from the Americas.

KPC-2-producing Klebsiella pneumoniae isolates mainly correspond to clonal complex 258 (CC258); however, we describe KPC-2-producing K. pneumoniae isolates belonging to invasive sequence type 23 (ST23). KPC-2 has scarcely been reported to occur in ST23, and this report describes the first isolation of this pathogen in the Americas. Acquisition of resistant markers in virulent clones could mark an evolutionary step toward the establishment of these clones as major nosocomial pathogens.

First detection of CMY-2 plasmid mediated ß-lactamase in Salmonella Heidelberg in South America.

Salmonella enterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries. Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the ß-lactam resistance profile. S. Heidelberg contained a 97kb plasmid belonging to the Inc N group harboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization. The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identified the first CMY-2 producing isolate of S. Heidelberg in Argentina and even in South America.

Genomic characterization of blaVIM-11-harbouring plasmids recovered from Pseudomonas aeruginosa.

OBJECTIVES: To the best of our knowledge, no genomic descriptions of blaVIM-11-harbouring plasmids are available in literature so far. The aim of this study was to describe the genomic features of three blaVIM-11-harbouring plasmids recovered from Pseudomonas aeruginosa isolated in Argentina in different periods. METHODS: blaVIM-11-harbouring plasmids from three clinical P. aeruginosa isolates were transferred by transformation into P. aeruginosa PAO-1. Then, genomic DNA of these transformants was extracted and sequenced using NovaSeq 6000 System-Illumina. De novo assemblies were generated using Unicycler program and reads were mapped against a reference genome of P. aeruginosa PAO-1. Plasmids sequences were predicted identifying the reads that did not map the reference sequence of PAO-1. These reads were recovered and assembled de novo. In silico predictions were carried out using bioinformatics tools. RESULTS: One Plasmid (pP6VIM-11) was distributed in 2 contigs, a second plasmid (pPOta2VIM-11) was found in a single contig, and the last one (pP936401VIM-11) was fragmented into 4 contigs. pP6VIM-11 and pPOta2VIM-11 belonged to the IncP-1ß group, displaying 64% of coverage and 83.9% of identity among them. pP936401VIM-1 plasmid corresponded to the IncN group. The bioinformatic analysis revealed that blaVIM-11 was located in a class 1 integron, flanked by insertion sequences, exhibiting potential for its dissemination. However, none of the plasmids were conjugative. CONCLUSION: This study corresponded to the first description and deposit of blaVIM-11-harbouring plasmids in P. aeruginosa, which expands the limited knowledge about their molecular epidemiology.

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae ST307 revealed multiple introductions in Buenos Aires, Argentina.

OBJECTIVES: To describe at genomic level nine carbapenemase-producing Klebsiella pneumoniae ST307 (Kp-ST307) clinical isolates recovered in Buenos Aires during 2017 to 2021, investigating their resistome, virulome, and phylogeny. METHODS: Antimicrobial susceptibility was determined according to Clinical and Laboratory Standards Intitute (CLSI). Genomic DNA was sequenced by Illumina MiSeq and analysed using SPAdes, PROKKA, and Kleborate. Phylogeny of 355 randomly selected Kp-ST307 genomes and those from nine local isolates was inferred by a maximum-likelihood approach. The tree was visualized using Microreact. RESULTS: Besides resistance to ß-lactams and fluoroquinolones, six out of nine Kp-ST307 were also resistant to ceftazidime/avibactam (CZA). This difficult-to-treat resvistance phenotype was mediated by blaSHV-28 and GyrA-83I/ParC-80I mutations in addition to carbapenemase coding genes. Among CZA susceptible isolates, two of them harboured blaKPC-3 while the other harboured blaKPC-2+blaCTX-M-15. Regarding CZA-resistant isolates, three harboured blaKPC-3+blaNDM-1+blaCMY-6, two carried blaKPC-2+blaNDM-5+blaCTX-M-15, and blaNDM-5+blaCTX-M-15 were detected in the remaining isolate. Furthermore, five colistin-resistant isolates presented a nonsense mutation in mgrB. Global Kp-ST307 isolates were distributed in two deep-branching lineages while local isolates were set in the main clade of the phylogenetic tree. The five isolates from the same hospital, harbouring blaKPC-3 or blaKPC-3+blaNDM-1+blaCMY-6, clustered in a monophyletic subclade with Italian isolates. Also, an isolate harbouring blaKPC-2+blaNDM-5+blaCTX-M-15 recovered in another hospital was closed to this group. The remaining local Kp-ST307 were grouped in other subclades containing isolates of diverse geographical origin. CONCLUSION: The inferred resistome was consistent with the resistant phenotype. Phylogeny suggested multiple introduction events in our region and a single major introduction in one hospital followed by local spread.

OXA-258 from Achromobacter ruhlandii: a species-specific marker.

A new blaOXA-258 gene is described as a species-specific taxonomic marker for Achromobacter ruhlandii isolates (all recovered from cystic fibrosis patients). Even though OXA-258 differs from OXA-114 variants, isolates could be misidentified as A. xiloxosidans by the amplification of an inner fragment from the OXA-coding gene. A robust identification of A. ruhlandii can be achieved by sequencing this single OXA gene, as well as by a more laborious recently proposed multilocus sequence-typing (MLST) scheme.

Promoting Science Literacy and Awareness across the Globe: the Role of Scientists as Science Ambassadors.

Science literacy has many personal and societal benefits that allows for better informed decision-making. Although the importance of science literacy is recognized globally, there are many challenges associated with its promotion. Scientists are more frequently engaging with nonscientific audiences through public outreach activities and with increasing support from institutions and professional societies. This is especially true regarding microbiologists and other related professionals since the start of the global 2019 coronavirus disease pandemic heightened the need to convey novel and rapidly evolving scientific information to lay audiences. The means by which professionals engage with these audiences affect the efficacy of the relay of scientific information. One method of engagement is the "ambassador approach," which aims to establish dialogue among different groups of people and scientists. In this perspective article, we discuss this approach, highlighting activities for the promotion of science literacy organized by the American Society for Microbiology Ambassador Program and similar programs of other scientific societies. We discuss the benefits and challenges of implementing an ambassador approach, propose potential improvements that could be made to existing programs promoting science literacy, and ultimately advocate for increased implementation of science ambassador programs.

First national survey of antibiotic susceptibility of the Bacteroides fragilis group: emerging resistance to carbapenems in Argentina.

The antibiotic susceptibility rates of 363 clinical Bacteroides fragilis group isolates collected from 17 centers in Argentina during the period from 2006 to 2009 were as follows: piperacillin-tazobactam, 99%; ampicillin-sulbactam, 92%; cefoxitin, 72%; tigecycline, 100%; moxifloxacin, 91%; and clindamycin, 52%. No metronidazole resistance was detected in these isolates during this time period. Resistance to imipenem, doripenem, and ertapenem was observed in 1.1%, 1.6%, and 2.3% of B. fragilis group strains, respectively. B. fragilis species showed a resistance profile of 1.5% to imipenem, 1.9% to doripenem, and 2.4% to ertapenem. This is the first report of carbapenem resistance in Argentina. The cfiA gene was present in 8 out of 23 isolates, all of them belonging to the B. fragilis species and displaying reduced susceptibility or resistance to carbapenems (MICs ≥ 4 µg/ml). Three out of eight cfiA-positive isolates were fully resistant to carbapenems, while 5 out of 8 isolates showed low-level resistance (MICs, 4 to 8 µg/ml). The inhibition by EDTA was a good predictor of the presence of metallo-ß-lactamases in the fully resistant B. fragilis strains, but discrepant results were observed for low-level resistant isolates. B. fragilis was more susceptible to antimicrobial agents than other Bacteroides species. Bacteroides vulgatus species was the most resistant to ampicillin-sulbactam and piperacillin-tazobactam, and B. thetaiotaomicron/ovatus strains showed the highest level of resistance to carbapenems, with an unknown resistance mechanism. B. vulgatus and the uncommon non-Bacteroides fragilis species were the most resistant to moxifloxacin, showing an overall resistance rate of 15.1%.

Plasmid-Encoded AmpC (pAmpC) in Enterobacteriaceae: epidemiology of microorganisms and resistance markers.

CMY-2 Β-lactamase is an important cause of Β-lactam resistance in Enterobacteriaceae and constitutes the most widespread pAmpC. Although CMY-2 has been previously recognized in our region, the real prevalence and epidemiology of this resistance marker was uncertain. During August-October 2009, we conducted a multicenter, prospective study to determine pAmpC prevalence and to characterize CMY-2 producing Escherichia coli associated plasmids. Plasmid-encoded AmpC prevalence was 0.9 % in enterobacteria in this period, being CMY-2 prevalent and to a lesser extent DHA. Molecular typing of CMY-2- producing Escherichia coli isolates showed several lineages. Moreover, replicon typing of cmy-2- containing plasmids displayed a broad diversity in Inc/cmy-2 links. Therefore, association of cmy-2 with specific transposon elements may be responsible for the spread of this resistance marker in Enterobacteriaceae.

Characterisation of blaKPC-2-harbouring plasmids recovered from Pseudomonas aeruginosa ST654 and ST235 high-risk clones.

OBJECTIVE: The main objectives were to describe two blaKPC-2 plasmids recovered from Pseudomonas aeruginosa isolates belonging to the ST654 and ST235 high-risk clones, and to compare with complete sequences of blaKPC-2 harbouring plasmids available in public databases. METHODS: Antimicrobial susceptibility was determined according to CLSI (Clinical and Laboratory Standards Institute) guidelines. Genomes were sequenced using an Illumina MiSeq platform, and blaKPC-2 plasmid sequences were achieved using MinION platform. Sequences were analysed using Unicycler and RAST. In silico predictions of the isolates sequence type (ST), antimicrobial resistance genes, plasmid replicon typing and MOB relaxases were fulfilled using bioinformatics tools. RESULTS: PA_2047 and PA_HdC isolates corresponded to the high-risk clones ST654 and ST235, respectively. The carbapenem resistance was mediated by KPC-2. Both blaKPC-2 harbouring plasmids, pPA_2047 and pPA_HdC, were different among them, non-conjugative and untypable by PlasmidFinder. pPA_2047 presented high identity with a Pae-13 plasmid, and these both located blaKPC-2 in Tn4401b isoform. pPA_HdC displayed a novel architecture, and the genetic context of blaKPC-2 was original. Besides the blaKPC-2 gene, resistance genes to aminoglycosides and quinolones were detected, including the novel phosphotransferase CrpP in PA_HdC. CONCLUSION: This study expands the limited knowledge about the molecular epidemiology of blaKPC-2 in P. aeruginosa from Latin America. Two novel plasmids harbouring blaKPC-2 were described that were untypable by their incompatibility group. The plasmid recovered from P. aeruginosa PA_HdC (ST235) displayed a novel architecture and an original context for blaKPC-2. On the other hand, the genetic platform carrying blaKPC-2 in P. aeruginosa PA_2047 (ST654) seems to a be a classical one.

Outbreak of Klebsiella pneumoniae ST11 Resistant To Ceftazidime-Avibactam Producing KPC-31 and the Novel Variant KPC-115 during COVID-19 Pandemic in Argentina.

We describe an outbreak of Klebsiella pneumoniae sequence type 11 (ST11) producing KPC variants resistant to ceftazidime-avibactam. Six patients hospitalized in the intensive care unit (mostly due to critical COVID pneumonia) presented infection or colonization by this bacterium. They had several comorbidities and required mechanical ventilation, central venous catheters, and urinary catheters. All 6 patients had a history of fecal colonization with KPC-producing Enterobacterales (KPC-E). Three of them had previous episodes of infection with ceftazidime-avibactam-susceptible KPC-producing K. pneumoniae, which were treated with ceftazidime-avibactam. Several phenotypic methods failed to detect carbapenemase production in these 6 ceftazidime-avibactam-resistant isolates, and they showed in vitro susceptibility to imipenem and meropenem. All of them rendered positive results for blaKPC by PCR, and amplicon sequencing identified blaKPC-31 variant in 5 isolates and a novel variant, named blaKPC-115, in the other. Moreover, matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to detect KPC in all isolates. Ceftazidime-avibactam-resistant isolates, as well as those recovered from previous infection episodes (KPC-3-producing K. pneumoniae, ceftazidime-avibactam susceptible), displayed a unique pulse type and belonged to ST11. Based on whole-genome sequencing results of selected isolates, less than 7 single-nucleotide polymorphisms were identified among them, which was indicative of the presence of a unique clone. Both in vivo selection and horizontal transmission seemed to have occurred in our hospital. Detection of these strains is challenging for the laboratory. History of previous KPC-E infections or colonization and systematic testing for resistance to ceftazidime-avibactam might help raise awareness of this possibility. IMPORTANCE Klebsiella pneumoniae is one of the main bacteria that cause infections in health care settings. This pathogen has developed a high level of resistance to many antibiotics. Some K. pneumoniae isolates can produce an enzyme known as carbapenemase KPC, making carbapenems (considered the last line for therapy) not effective to treat their infections. The combination ceftazidime-avibactam, approved by FDA in 2015, is useful to treat infections caused by KPC-producing K. pneumoniae. This study describes the emergence, in one hospital in Argentina, of K. pneumoniae isolates that produce KPC variants (KPC-31 and KPC-115) resistant to ceftazidime-avibactam. The ceftazidime-avibactam-resistant bacteria were isolated in inpatients, including some that previously received this combination as treatment. Transmission of this strain to other patients also occurred in the studied period. Detection of these bacteria is challenging for the laboratory. The knowledge and awareness of the emergence of this pathogen in our region are highly valuable.