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.

MALDI-TOF MS-Based KPC Direct Detection from Patients' Positive Blood Culture Bottles, Short-Term Cultures, and Colonies at the Hospital.

Carbapenemase resistance in Enterobacterales is a global public health problem and rapid and effective methods for detecting these resistance mechanisms are needed urgently. Our aim was to evaluate the performance of a MALDI-TOF MS-based "Klebsiella pneumoniae carbapenemase" (KPC) detection protocol from patients' positive blood cultures, short-term cultures, and colonies in healthcare settings. Bacterial identification and KPC detection were achieved after protein extraction with organic solvents and target spot loading with suitable organic matrices. The confirmation of KPC production was performed using susceptibility tests and blaKPC amplification using PCR and sequencing. The KPC direct detection (KPC peak at approximately 28.681 Da) from patients' positive blood cultures, short-term cultures, and colonies, once bacterial identification was achieved, showed an overall sensibility and specificity of 100% (CI95: [95%, 100%] and CI95: [99%, 100%], respectively). The concordance between hospital routine bacterial identification protocol and identification using this new methodology from the same extract used for KPC detection was ≥92%. This study represents the pioneering effort to directly detect KPC using MALDI-TOF MS technology, conducted on patient-derived samples obtained from hospitals for validation purposes, in a multi-resistance global context that requires concrete actions to preserve the available therapeutic options and reduce the spread of antibiotic resistance markers.

Comparative Genomics Identifies Novel Genetic Changes Associated with Oxacillin, Vancomycin and Daptomycin Susceptibility in ST100 Methicillin-Resistant Staphylococcus aureus.

Infections due to vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) represent a serious concern due to their association with vancomycin treatment failure. However, the underlying molecular mechanism responsible for the hVISA/VISA phenotype is complex and not yet fully understood. We have previously characterized two ST100-MRSA-hVISA clinical isolates recovered before and after 40 days of vancomycin treatment (D1 and D2, respectively) and two in vitro VISA derivatives (D23C9 and D2P11), selected independently from D2 in the presence of vancomycin. This follow-up study was aimed at further characterizing these isogenic strains and obtaining their whole genome sequences to unravel changes associated with antibiotic resistance. It is interesting to note that none of these isogenic strains carry SNPs in the regulatory operons vraUTSR, walKR and/or graXRS. Nonetheless, genetic changes including SNPs, INDELs and IS256 genomic insertions/rearrangements were found both in in vivo and in vitro vancomycin-selected strains. Some were found in the downstream target genes of the aforementioned regulatory operons, which are involved in cell wall and phosphate metabolism, staphylococcal growth and biofilm formation. Some of the genetic changes reported herein have not been previously associated with vancomycin, daptomycin and/or oxacillin resistance in S. aureus.

Emergence of Urease-Negative Klebsiella pneumoniae ST340 Carrying an IncP6 Plasmid-Mediated blaKPC-2 Gene.

An unusual biotype of KPC-2-producing Klebsiella pneumoniae (KPC-Kpn) isolates was detected in Corrientes, Argentina, which, to their isolation date, had been free of KPC-Kpn outbreaks. Our aim was to describe the clinical epidemiology focused on genomic characterization of atypical urease-negative KPC-Kpn clinical isolates belonging to the high-risk hospital-associated clonal lineage ST340/CC258. Thirteen isolates were recovered, all of them from inpatients with KPC-Kpn infection (August 2015 to January 2016). These isolates displayed identical enterobacterial repetitive intergenic consensus-PCR electropherotype belonging to a single clonal sequence type ST340. Whole genome sequencing was performed on two KPC-Kpn and the resistome analyses revealed the following acquired resistance genes: blaKPC-2, blaCTX-M-15, blaOXA-1, blaSHV-11, aac(3)-IId, aph(3')-Ia, aac(6')-Ib-cr, sul1, dfrA14, catB3, fosA, and arr-3. Mutations in GyrA (S83I) and ParC (S80I) were also identified. Among the virulence determinants, yersiniabactin was detected in both strains, specifically the ybt9 locus located in ICEKp3. Five plasmid incompatibility groups were observed in this clone and an unusual IncP6 plasmid bearing blaKPC-2 gene (named pKpn3KP) was fully characterized. In this study, we present the first draft genome sequences of two clinical isolates of KPC-2/CTX-M-15-producing K. pneumoniae belonging to the high-risk clonal lineage ST340/CC258 associated with nosocomial outbreaks in Argentina.

Detecting KPC-2 and NDM-1 Coexpression in Klebsiella pneumoniae Complex from Human and Animal Hosts in South America.

Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-ß-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for "CZA 50" (30 µg ceftazidime [CAZ] and 20 µg AVI) and "CZA 14" (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide. IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.

Whole-Genome Analysis of a High-Risk Clone of Klebsiella pneumoniae ST147 Carrying Both mcr-1 and blaNDM-1 Genes in Peru.

The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other ß-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

AmpR is a dual regulator in Stenotrophomonas maltophilia with a positive role in ß-lactam resistance and a negative role in virulence, biofilm and DSF production.

Stenotrophomonas maltophilia intrinsic resistance to ß-lactams is mediated by two chromosomal ß-lactamases, L1 and L2, whose induction depends on AmpR. Its quorum sensing (QS) signal, the diffusible signal factor (DSF), has a positive role in biofilm production, virulence and induction of ß-lactamases. We hypothesized that AmpR has a role in virulence, biofilm production and QS system. Studies were done on S. maltophilia K279a, K279a ampRFS (ampR deficient mutant) and K279aM11 (constitutively active AmpR mutant). K279a ampRFS showed the highest biofilm biomass, thickness and 3D organization. Conversely, K279aM11 was the least efficient biofilm former strain. qRT-PCR showed that spgM, related to biofilm formation and virulence, was upregulated in K279a ampRFS and downregulated in K279aM11. A constitutively active AmpR led to a reduction of DSF production, while K279a ampRFS was the highest producer. Consequently, qRT-PCR showed that AmpR negatively regulated rpfF expression. K279a ampRFS presented the highest oxidative stress resistance, overexpressed sodA gene and showed the highest virulence in the Galleria mellonella killing assay. This is the first evidence of the function of AmpR as a dual regulator in S. maltophilia with a positive role in ß-lactam resistance and a negative role in DSF production, biofilm formation, oxidative stress resistance and virulence.

Characterization of Emerging Pathogens Carrying blaKPC-2 Gene in IncP-6 Plasmids Isolated From Urban Sewage in Argentina.

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all ß-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, blaKPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring blaKPC-2. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by ΔISKpn6/blaKPC-2/ΔblaTEM-1/ISKpn27. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting blaKPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of blaKPC-2 in the environment.

Molecular epidemiology of cefotaxime-resistant but ceftazidime-susceptible Enterobacterales and evaluation of the in vitro bactericidal activity of ceftazidime and cefepime.

Extended-spectrum ß-lactamases' (ESBLs) production is the main resistance mechanism to third-generation cephalosporins (TGCs) in gram-negative bacilli. In Argentina, there is a high prevalence of cefotaximase-type ESBLs (CTX-M). For this reason, dissociated resistance phenotype (DRP) displaying a profile of resistance to cefotaxime (CTX) and susceptibility to ceftazidime (CAZ) might be detected. The aims of this study were to determine the prevalence of DRP in Enterobacterales clinical isolates, to characterize the mechanisms responsible for this phenotype and to evaluate the in vitro behaviour against different antibiotics. Sixty Enterobacterales resistant to any TGC were studied, and among them, 25% displayed a DRP. The ß-lactamases associated with DRP were 5/11 CTX-M-2, 4/11 CTX-M-14, 1/11 CTX-M-15 and 1/11 CMY-2 in E. coli, 2/3 CTX-M-2 and 1/3 CMY-2 in P. mirabilis and 1/1 CTX-M-14 in K. pneumoniae. Furthermore, CTX-M-2 and CTX-M-14 were related with DRP in both wild-type isolates and the corresponding transconjugants. Time-kill experiments showed CAZ bactericidal activity on CTX-M-2-and CTX-M-14-producing strains and bacterial regrowth in those CMY-2 producers. An opposite behaviour was evident when cefepime (FEP) was used. However, CAZ and gentamicin combination showed a synergistic effect against the CMY-2 producers. We concluded that Enterobacterales with DRP responded differently to CAZ or FEP depending on the type of ß-lactamase they possess, suggesting that these cephalosporins could be a therapeutic option. Therefore, the characterization of the involved resistance mechanism might contribute to define the appropriate antibiotic treatment.

Co-Occurrence of NDM-5 and RmtB in a Clinical Isolate of Escherichia coli Belonging to CC354 in Latin America.

New Delhi metallo-ß-lactamase (NDM)-producing isolates are usually resistant to most ß-lactams and other antibiotics as a result of the coexistence of several resistance markers, and they cause a variety of infections associated to high mortality rates. Although NDM-1 is the most prevalent one, other variants are increasing their frequency worldwide. In this study we describe the first clinical isolate of NDM-5- and RmtB-producing Escherichia coli in Latin America. E. coli (Ec265) was recovered from a urine sample of a female outpatient. Phenotypical and genotypical characterization of resistance markers and conjugation assays were performed. Genetic analysis of Ec265 was achieved by whole genome sequencing. Ec265 belonging to ST9693 (CC354), displayed resistance to most ß-lactams (including carbapenems), aminoglycosides (gentamicin and amikacin), and quinolones. Several resistance genes were found, including blaNDM-5 and rmtB, located on a conjugative plasmid. blaNDM-5 genetic context is similar to others found around the world. Co-transfer of multiple antimicrobial resistance genes represents a particular challenge for treatment in clinical settings, whereas the spread of pathogens resistant to last resort antibiotics should raise an alarm in the healthcare system worldwide.

Antimicrobial resistance in bacterial isolates from companion animals in Buenos Aires, Argentina: 2011-2017 retrospective study.

The widespread use of antimicrobial therapy in companion animals could lead to increased levels of resistance. Monitoring these levels is critical to understand not only the zoonotic threat of resistant bacteria but also the interspecies transmission of resistance mechanisms. However, data on resistance levels in companion animals of urban areas in Latin America are lacking. In this retrospective study, we analysed 23,922 isolates recovered from clinical samples of dogs and cats between 2011 and 2017. Growing trends of resistance to fluoroquinolones were observed in most bacterial species of veterinary importance with zoonotic potential (Enterobacterales, Pseudomonas and Staphylococcus). Furthermore, an increase of resistance levels to third-generation cephalosporins was also detected in Enterobacterales. Currently, Pseudomonas aeruginosa, Enterococcus spp. and Streptococcus spp. did not seem to represent a clinical challenge. A high proportion of multidrug-resistant (MDR) Enterobacterales isolated from urine was reported. It is interesting to outline that resistance to amikacin was exceptional. This study might be considered as a baseline for prospective antimicrobial resistance surveillance in companion animals in our region.

Dissemination of bla NDM- 1 Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.

Nosocomial infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are a major health problem worldwide. The aim of this study was to describe NDM-1-producing K. pneumoniae strains causing bacteremia in a tertiary referral hospital in Mexico. MDR K. pneumoniae isolates were screened by polymerase chain reaction for the presence of resistance genes. In resistant isolates, plasmids were identified and conjugation assays were performed. Clonal diversity and the sequence types were determined by pulsed-field gel electrophoresis and multilocus sequence typing. A total of 80 K. pneumoniae isolates were collected from patients with bacteremia over a 1-year period. These isolates showed a level of resistance of 59% (47/80) to aztreonam, 56-60% (45-48/80) to cephalosporins, 54% (43/80) to colistin and 12.5% (10/80) to carbapenems. The carbapenem resistant isolates were bla NDM- 1 carriers and negative for bla KPC, bla NDM, bla IMP, bla VIM and bla OXA- 48 -like carbapenemases genes. Conjugative plasmids IncFIIA and IncF group with sizes of 82-195 kbp were carriers of bla NDM- 1, bla CTX-M- 15, bla TEM- 1, aac(6')-Ib and/or aac(3')-IIa. Clonal variability and nine different multilocus sequence types were detected (ST661, ST683, ST1395, ST2706, ST252, ST1198, ST690, ST1535, and ST3368) for the first time in the isolates carrying bla NDM- 1 in Mexico. This study demonstrates that bla NDM- 1 has remained within this hospital in recent years and suggests that it is currently the most prevalent carbapenemase among K. pneumoniae MDR strains causing bacteremia in Mexico. The horizontal transfer of bla NDM- 1 gene through IncF-like plasmids among different clones demonstrates the dissemination pathway of antimicrobial resistance and underscore the need for strong and urgent joint measures to control the spread of NDM-1 carbapenemase in the hospital.

Genetic changes associated with tigecycline resistance in Staphylococcus aureus in vitro-selected mutants belonging to different lineages.

Tigecycline (TGC) resistance remains rare in Staphylococcus aureus worldwide. In this study, 12 TGC-resistant S. aureus mutants (TRSAm) were obtained displaying an increase in efflux activity. The isolates belonged to seven different genetic lineages, with a predominance of clonal complex 5 (CC5). Diverse genetic changes in mepA and mepR genes were found producing alterations in the amino acid sequences of the corresponding proteins (MepA and MepR, respectively). The most frequent amino acid change in MepA was Glu287Gly. All of the TRSAm exhibited different single nucleotide polymorphisms (SNPs) or insertions/deletions (InDels) in mepR causing premature stop codons or amino acid changes in MepR. Expression of mepA was significantly increased in TRSAm with different mutations in mepA and mepR. Of the 12 TRSAm, 6 also harboured mutations in rpsJ that resulted in amino acid changes in the S10 ribosomal protein, with Lys57 being the most frequently mutated site. Our findings demonstrate that these acquired mechanisms of TGC resistance are not restricted to a single type of genotypic background and that different lineages might have the same plasticity to develop TGC resistance. The impact of TGC selective pressure assessed by whole-genome sequencing in four selected strain pairs revealed mutations in other singular genes and IS256 mobilisation.

Comparative genomics of ST5 and ST30 methicillin-resistant Staphylococcus aureus sequential isolates recovered from paediatric patients with cystic fibrosis.

Staphylococcus aureus chronic airway infection in patients with cystic fibrosis (CF) allows this pathogen to adapt over time in response to different selection pressures. We have previously shown that the main sequence types related to community-acquired methicillin-resistant S. aureus (MRSA) infections in Argentina - ST5 and ST30 - are also frequently isolated from the sputum of patients with CF, but in these patients they usually display multi-drug antimicrobial resistance. In this study, we sequenced the genomes of MRSA from four paediatric CF patients with the goal of identifying mutations among sequential isolates, especially those possibly related to antimicrobial resistance and virulence, which might contribute to the adaptation of the pathogen in the airways of patients with CF. Our results revealed genetic differences in sequential MRSA strains isolated from patients with CF in both their core and accessory genomes. Although the genetic adaptation of S. aureus was distinct in different hosts, we detected independent mutations in thyA, htrA, rpsJ and gyrA - which are known to have crucial roles in S. aureus virulence and antimicrobial resistance - in isolates recovered from multiple patients. Moreover, we identified allelic variants that were detected in all of the isolates recovered after a certain time point; these non-synonymous mutations were in genes associated with antimicrobial resistance, virulence, iron scavenging and oxidative stress resistance. In conclusion, our results provide evidence of genetic variability among sequential MRSA isolates that could be implicated in the adaptation of these strains during chronic CF airway infection.

Molecular characterization and antimicrobial resistance profiles of Salmonella Heidelberg isolates from poultry.

In the last 10 years, Salmonella Heidelberg has been extensively isolated from poultry in several countries. In this context, molecular characterization is essential to understand whether the strains have entered the farms from a single or several sources. Thus, the aim of this study was to determine the genetic relationship and antimicrobial susceptibility of S. Heidelberg strains isolated between 2011 and 2012 from broiler farms belonging to three integrated poultry companies located in Argentina. The genetic relatedness of the S. Heidelberg isolates was determined by pulsed-field gel electrophoresis (PFGE), and resistance to 21 antimicrobials was determined by the disc diffusion method. The isolates were assigned to four PFGE patterns. Most of the strains showed 100% similarity and belonged to the same integrated poultry company. This PFGE pattern was also prevalent in S. Heidelberg strains isolated from humans in several provinces of Argentina, which suggests an epidemiological association between human and poultry strains. All the isolates were classified as multidrug-resistant (MDR), and no clear relationship was observed between PFGE and resistance patterns. S. Heidelberg strains may circulate among farms from the same integrated company due to common sources of contamination. To guarantee the safety of the poultry product for the consumers, holistic approaches including surveillance of Salmonella throughout the production chain together with control measures are crucial.

Stenotrophomonas maltophilia phenotypic and genotypic features through 4-year cystic fibrosis lung colonization.

Introduction. Stenotrophomonas maltophilia has emerged as one of the most common multi-drug-resistant pathogens isolated from people with cystic fibrosis (CF). However, its adaptation over time to CF lungs has not been fully established.Hypothesis. Sequential isolates of S. maltophilia from a Brazilian adult patient are clonally related and show a pattern of adaptation by loss of virulence factors.Aim. To investigate antimicrobial susceptibility, clonal relatedness, mutation frequency, quorum sensing (QS) and selected virulence factors in sequential S. maltophilia isolates from a Brazilian adult patient attending a CF referral centre in Buenos Aires, Argentina, between May 2014 and May 2018.Methodology. The antibiotic resistance of 11 S. maltophilia isolates recovered from expectorations of an adult female with CF was determined. Clonal relatedness, mutation frequency, QS variants (RpfC-RpfF), QS autoinducer (DSF) and virulence factors were investigated in eight viable isolates.Results. Seven S. maltophilia isolates were resistant to trimethoprim-sulfamethoxazole and five to levofloxacin. All isolates were susceptible to minocycline. Strong, weak and normomutators were detected, with a tendency to decreased mutation rate over time. XbaI PFGE revealed that seven isolates belong to two related clones. All isolates were RpfC-RpfF1 variants and DSF producers. Only two isolates produced weak biofilms, but none displayed swimming or twitching motility. Four isolates showed proteolytic activity and amplified stmPr1 and stmPr2 genes. Only the first three isolates were siderophore producers. Four isolates showed high resistance to oxidative stress, while the last four showed moderate resistance.Conclusion. The present study shows the long-time persistence of two related S. maltophilia clones in an adult female with CF. During the adaptation of the prevalent clones to the CF lungs over time, we identified a gradual loss of virulence factors that could be associated with the high amounts of DSF produced by the evolved isolates. Further, a decreased mutation rate was observed in the late isolates. The role of all these adaptations over time remains to be elucidated from a clinical perspective, probably focusing on the damage they can cause to CF lungs.

Expansion and improvement of MALDI-TOF MS databases for accurate identification of Achromobacter species.

Different MALDI-TOF MS databases were evaluated for the identification of Achromobacter species. The in-house and extended database generated in this study rendered more accurate identification (58/64 and 57/64 isolates, respectively) in comparison with the Bruker commercial database (42/64 isolates), especially in those infrequent species that are not available or poorly represented.

Bacteremia and sepsis by Arcanobacterium haemolyticum in a young immunocompetent patient.

We report the case of a twenty-year-old immunocompetent male patient presenting to the emergency room with pharyngitis and fever. Blood cultures were drawn and Arcanobacterium haemolyticum (rough biotype) was recovered. The presence of the arcanolysin gene was investigated at the molecular level and the upstream region was amplified and sequenced in order to correlate it with the smooth or rough biotype. Although the isolate was susceptible to penicillin, vancomycin and gentamicin, empirical treatments first with amoxicillin/clavulanic acid (1g/12h) and then with ceftriaxone (1g/12h) failed and the infection evolved to sepsis. Finally, treatment with vancomycin (1g/12h) plus piperacillin/tazobactam (4.5g/8h) was effective. Lemierre's syndrome was ruled out. To the best of our knowledge, this is the first case of bacteremia by A. haemolyticum reported in Argentina.