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Background Lateral flow immunoassays (LFIA) have shown their usefulness for detecting CTX-M- and carbapenemase-producing Enterobacterales (CPEs) in bacterial cultures. Here, we have developed and validated the BL-DetecTool to detect CTX-M enzymes and carbapenemases directly from clinical samples. Methods The BL-DetecTool is an LFIA that integrates an easy sample preparation device named SPID (Sampling, Processing, Incubation and Detection). It was evaluated in three University hospitals on urine, blood culture (BC) and rectal swab (RS) specimens either of clinical origin or on spiked samples. RS evaluation was done directly and after a 24 h enrichment step. Results The CTX-M BL-DetecTool was tested on 485 samples (154 BC, 150 urines, and 181 RS) and revealed a sensitivity and specificity of 97.04% (95% CI 92.59%–99.19%) and 99.43% (95% CI 97.95%–99.93%), respectively. Similarly, the Carba5 BL-DetecTool was tested on 382 samples (145 BC, 116 urines, and 121 RS) and revealed a sensitivity and specificity of 95.3% (95% CI 89.43%–98.47%) and 100% (95% CI 98.67%–100%), respectively. While with the Carba5 BL-DetecTool five false negatives were observed, mostly in RS samples, with the CTX-M BL-DetecTool, in addition to four false-negatives, two false-positives were also observed. Direct testing of RS samples revealed a sensitivity of 78% and 86% for CTX-M and carbapenemase detection, respectively. Conclusions BL-DetecTool showed excellent biological performance, was easy-to-use, rapid, and could be implemented in any microbiology laboratory around the world, without additional equipment, no need for electricity, nor trained personnel. It offers an attractive alternative to costly molecular methods.

Objectives Protegrins are a family of natural peptides from the innate immune system of vertebrates, with broad-spectrum antimicrobial activity. However, the toxicity and haemolysis of protegrin-1 (PG-1) at low concentrations renders it useless for therapeutic application. We rationally designed PLP-3, a novel synthetic PG-1-like peptide, comprising key activity features of protegrins in a constrained bicyclic structure. Our main objective was to investigate PLP-3’s activity against MDR strains of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae and to analyse its haemolysis and cytotoxicity. Methods Peptide synthesis was performed via solid phase and intramolecular ligation in solution, and the correct folding of the peptide was verified by circular dichroism. Antimicrobial activity was performed through broth microdilution. The test panel contained 45 bacterial strains belonging to A. baumannii, P. aeruginosa and K. pneumoniae (15 strains per species) comprising colistin-resistant and MDR strains. Cytotoxicity was assessed by XTT cell viability assays using HeLa and A549 cells and haemolysis of human erythrocytes. Results PLP-3 was successfully synthesized, and its antiparallel β-sheet conformation was confirmed. Antimicrobial activity screening showed MIC90 values of 2 mg/L for A. baumannii, 16 mg/L for K. pneumoniae and 8 mg/L for P. aeruginosa. The haemolysis IC50 value was 48.53 mg/L. Cytotoxicity against human HeLa and A549 cells showed values of ca. 200 mg/L in both cell lines resulting in a 100-fold selectivity window for bacterial over human cells. Conclusions PLP-3 has potent antimicrobial activity, especially against A. baumannii, while maintaining low haemolysis and toxicity against human cell lines at antimicrobial concentrations. These characteristics make PLP-3 a promising peptide with an interesting therapeutic window.

Cefiderocol is a catechol-substituted siderophore cephalosporin combining rapid penetration into the periplasmic space with increased stability against β-lactamases. This study provides additional data on the in vitro antimicrobial activity of cefiderocol and commercially available comparators against an epidemiologically diverse collection of

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P Salmonella spp. (P Campylobacter spp. (P G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

Background Minimizing the effect of antimicrobial resistance (AMR) requires an adequate policy response that relies on good governance and coordination. We have previously demonstrated a knowledge gap on infectious diseases within the general public, but equivalent data from a policy context are still lacking. The aim of this study is to have a better comprehension of how AMR is understood and perceived among policy makers and stakeholders in tackling AMR on a global level. Methods A digital survey was designed to capture the knowledge, attitude, and practices (KAP) towards AMR. A self-reporting questionnaire was distributed to politicians, policy advisors and relevant stakeholders, and survey responses were collected between November 2020 and March 2021. Results A total of 351 individuals participated in this study with 80% from high-income countries (HIC) and 20% from low- and middle-income countries (LMIC). Fifteen different countries were represented, and the majority of participants were from the Netherlands, Spain and Myanmar. Overall, participants had sufficient knowledge regarding AMR and reported the importance of political willingness in tackling AMR. More than half (65%) of participants from LMIC reported antibiotics misuse, and almost half (48%) claimed to be unaware of this health problem. Politicians and stakeholders from LMIC demonstrated a higher knowledge on AMR but showed poor perception and attitude toward antimicrobial use compared to those from HIC regions. HIC participants emphasized the role of the veterinary sector in particular, as well as the environmental dissemination of antibiotics and drug residues. The lack of funding resources was especially reported by participants from LMIC. Conclusion Inter-regional differences in KAP regarding AMR exist among politicians, policy advisors and relevant stakeholders. Overall, participants demonstrated to have a sufficient level of knowledge and awareness of AMR. This study characterizes a multi-national policymaker and stakeholder mapping that can be used to propose further policy implementation on various governance levels.

The introduction of a novel tetra‐ortho‐chloroazobenzene amino acid (CEBA) has enabled photoswitching of the antimicrobial activity of tyrocidine A analogues by using exclusively visible light, granting spatiotemporal control under benign conditions. Compounds bearing this photoswitchable amino acid become active upon irradiation with red light, but quickly turn‐off upon exposure to other visible light wavelengths. Critically, sunlight quickly triggers isomerisation of the red light‐activated compounds into their original trans form, offering an ideal platform for self‐deactivation upon release into the environment. Linear analogues of tyrocidine A were found to provide the best photocontrol of their antimicrobial activity, leading to compounds active against Acinetobacter baumannii upon isomerisation. Exploration of their N‐ and C‐termini has provided insights into key elements of their structure and has allowed obtaining new antimicrobials displaying excellent strain selectivity and photocontrol., A novel photoswitchable amino acid has provided access to tyrocidine A analogues fully operated under visible light. This has led to linear analogues that have shown good photoswitchability and strain selectivity against A. baumannii and S. pyogenes clinical strains by using different analogues. Activated cis compounds turn back to their deactivated trans form upon short exposure times to sunlight, paving the way to self‐deactivating antibacterials with minor evolutionary pressure on the environment.

Infectious diseases are emerging and re-emerging due to climate change. Understanding how climate variability affects the transmission of infectious diseases is important for both researchers and the general public. Yet, the widespread knowledge of the general public on this matter is unknown, and quantitative research is still lacking. A survey was designed to assess the knowledge and perception of 1) infectious diseases, 2) climate change and 3) the effect of climate change on infectious diseases. Participants were recruited via convenience sampling, and an anonymous cross-sectional survey with informed consent was distributed to each participant. Descriptive and inferential analyses were performed primarily focusing on the occupational background as well as nationality of participants. A total of 458 individuals participated in this study, and most participants were originally from Myanmar, the Netherlands, Spain, United Kingdom and the United States. Almost half (44%) had a background in natural sciences and had a higher level of knowledge on infectious diseases compared to participants with non-science background (mean score of 12.5 and 11.2 out of 20, respectively). The knowledge of the effect of climate change on infectious diseases was also significantly different between participants with and without a background in natural sciences (13.1 and 11.8 out of 20, respectively). The level of knowledge on various topics was highly correlated with nationality but not associated with age. The general population demonstrated a high awareness and strong knowledge of climate change regardless of their background in natural sciences. This study exposes a knowledge gap in the general public regarding the effect of climate change on infectious diseases, and highlights that different levels of knowledge are observed in groups with differing occupations and nationalities. These results may help to develop awareness interventions for the general public.

Background: Standard treatments against bacterial infections are becoming ineffective due to the rise of antibacterial resistance worldwide. Classical approaches to develop new antibacterial agents are not sufficient to fulfil the current pipeline, therefore new strategies are currently being devised in the field of antibacterial discovery. Objectives: The objective of this narrative review is to compile the most successful strategies for drug discovery within the antibacterial context that are currently being pursued. Sources: Peer-reviewed publications from the MEDLINE database with robust data addressing the discovery of new antibacterial agents in the current pipeline have been selected. Content: Several strategies to discover new antibacterials are described in this review: (i) derivatives of known antibacterial agents; the activity of a known antimicrobial agent can be improved through two strategies: (a) the modification of the original chemical structure of an antimicrobial agent to circumvent antibacterial resistance mechanisms and (b) the development of a compound that inhibits the mechanisms of resistance to an antibacterial agent; (ii) new antibacterial agents targeting new proteins; (iii) inhibitors of virulence factors; (iv) nanoparticles; (v) antimicrobial peptides and peptidomimetics; (vi) phage therapy and enzybiotics; and (vii) antisense oligonucleotides. Implications: This review intends to provide a positive message affirming that several different strategies to design new antibacterial agents are currently being developed, and we are therefore confident that in the near future some of the most promising approaches will come to fruition.

- b: - "Introduction:" - "Methods:" - "Ethical considerations:" sup: - th - th i: - ad hoc content: - " The Informed Health Choices (IHC) project has developed learning resources to teach primary school children (10 to 12-year-olds) to assess treatment claims and make informed health choices. The aim of our study is to explore both the students' and teachers' experience when using these resources in the context of Barcelona (Spain). " - " During the 2019-2020 school year, we will conduct a pilot study with 4 " - " and 5 " - "-year primary school students (9 to 11-year-olds) from three schools in Barcelona. The intervention in the schools will include: 1) assessment of the IHC resources by the teachers before the lessons, 2) non-participatory observations during the lessons, 3) semi-structured interviews with the students after a lesson, 4) assessment of the lessons by the teachers after a lesson, 5) treatment claim assessment by the students at the end of the lessons, and 6) assessment of the IHC resources by the teachers at the end of the lessons. We will use " - " questionnaires and guides to register the data. We will perform a quantitative and qualitative analysis of the data to explore understandability, desirability, suitability, usefulness, facilitators and barriers of the resources. The most relevant results will be discussed and some recommendations on how to use, how to adapt (if needed), and how to implement the IHC resources to this context will be agreed. The findings of the contextualization activities could inform the design of a cluster-randomised trial, to determine the effectiveness of the IHC resources in this context prior to scaling-up its use. " - " The study protocol has obtained an approval exemption from the Ethics Committee of the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain)."

Objectives During the period from December 2007 to November 2012, the epidemiology of diarrhea caused by Shigella was studied among children

Delfino Vubil,1 Sozinho Acácio,1,2 Llorenç Quintò,3 Clara Ballesté-Delpierre,3 Tacilta Nhampossa,1,2 Karen Kotloff,4 Myron M Levine,4 Pedro Alonso,1 James P Nataro,5 Tamer H Farag,4 Jordi Vila,3,6 Inacio Mandomando1,2 1Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique; 2Instituto Nacional de Saúde (INS), Maputo, Mozambique; 3ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain; 4Center for Vaccine Development (CVD), University of Maryland, School of Medicine, Baltimore, MD, USA; 5Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA, USA; 6Department of Clinical Microbiology, Centre for Biomedical Diagnosis, Hospital Clinic, Barcelona, Spain Objectives: During the period from December 2007 to November 2012, the epidemiology of diarrhea caused by Shigella was studied among children

OBJECTIVES The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents. METHODS PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT-PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing. RESULTS We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam. CONCLUSIONS Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the β-lactam least associated with the selection of resistance mechanisms.

Salmonella possesses virulence determinants that allow replication under extreme conditions and invasion of host cells, causing disease. Here, we examined four putative genes predicted to encode membrane proteins (ydiY, ybdJ, STM1441 and ynaJ) and a putative transcriptional factor (yedF). These genes were identified in a previous study of a S. Typhimurium clinical isolate and its multidrug-resistant counterpart. For STM1441 and yedF a reduced ability to interact with HeLa cells was observed in the knock-out mutants, but an increase in this ability was absent when these genes were overexpressed, except for yedF which phenotype was rescued when yedF was restored. In the absence of yedF, decreased expression was seen for: i) virulence-related genes involved in motility, chemotaxis, attachment and survival inside the host cell; ii) global regulators of the invasion process (hilA, hilC and hilD); and iii) factors involved in LPS biosynthesis. In contrast, an increased expression was observed for anaerobic metabolism genes. We propose yedF is involved in the regulation of Salmonella pathogenesis and contributes to the activation of the virulence machinery. Moreover, we propose that, when oxygen is available, yedF contributes sustained repression of the anaerobic pathway. Therefore, we recommend this gene be named vrf, for virulence-related factor.

The objective of this study was to assess antibiotic resistance and the molecular epidemiology of shigella isolates from a case-control study of diarrhoea, conducted from 2007 to 2012 in children aged less than 5 years in Manhica district, southern Mozambique. All isolates were tested for antimicrobial susceptibility using the disc diffusion method. Polymerase chain reaction was used to detect different molecular mechanisms of antibiotic resistance. Serotyping was performed using specific antisera. The clonal relationship of Shigella flexneri and Shigella sonnei was assessed by pulsed-field gel electrophoresis (PFGE). Of the 67 shigella isolates analysed, 59 were diarrhoeal cases and eight were controls. S. flexneri (70.1%; 47/67) was the most common species, followed by S. sonnei (23.9%; 16/67). The most prevalent S. flexneri serotypes were 2a (38.3%; 18/47), 6 (19.2%; 9/47) and 1b (14.9%; 7/47). High rates of antimicrobial resistance were observed for trimethoprim-sulfametoxazole (92.5%; 62/67), tetracycline (68.7%; 46/67), chloramphenicol (53.7%; 36/67) and ampicillin (50.7%; 34/67). Multi-drug resistance (MDR) was present in 55.2% (37/67) of the isolates and was associated with a case fatality rate of 8.1% (3/37). PFGE revealed 22 clones (16 S. flexneri and 6 S. sonnei), among which P1 (31.9%; 15/47), P9 (17%; 8/47) and P2 (10.6%; 5/47) were the most prevalent clones of S. flexneri. In conclusion, S. flexneri was the most prevalent species, with MDR isolates mainly belonging to three specific clones (P1, P9 and P2). The case fatality rate observed among MDR isolates is a matter of concern, indicating the need for appropriate treatment.

In the last few years, the number of Salmonella enterica strains resistant to nalidixic acid has steadily increased. In a previous study, the quinolone susceptibility phenotype and genotype of 38 S. enterica clinical isolates (19 S. enterica serovar Typhimurium and 19 S. enterica serovar Enteritidis) were determined. Forty-two percent of the isolates showed nalidixic acid resistance associated with a mutation in gyrA together with putative overexpression of efflux pump(s). In this study, mutations in the quinolone resistance-determining region (QRDR) of parE and the regulators of AcrAB (acrR, marRAB, soxRS and ramR) were analysed. Intracellular accumulation of ciprofloxacin and nalidixic acid was determined. Gene expression of the efflux pump components acrB, tolC, acrF and emrB was also assessed. In addition, an epidemiological study of the isolates by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) was performed. No mutations were detected in parE, whereas two amino acid substitutions were found in two susceptible strains in MarR (I84L) and AcrR (N214T) in one strain each, although both were suggested to be polymorphisms. No changes in the gene expression of acrB, tolC, acrF and emrB were detected between nalidixic-acid-resistant and -susceptible strains. Intracellular accumulation was not useful to reveal differences. Epidemiological analysis showed an important clonal relatedness among the S. Enteritidis isolates, whereas major divergence was seen for S. Typhimurium. Altogether, these results suggest the presence of previously undiscovered drug efflux pump(s) and confirm the high clonality of S. Enteritidis and the genetic divergence of S. Typhimurium.

Outer membrane proteins (OMPs) play an important role in the interaction of bacterial pathogens with host cells. Indeed, some OMPs from different Gram-negative bacteria have been recognized as important virulence factors for host immune recognition. This scenario has led to the study of the outer membrane (OM) subproteome of pathogenic bacteria as an essential step for gaining insight into the mechanisms of pathogenesis and for the identification of virulence factors. Although progress in the characterization of the OM has recently been reported, detailed protein composition of this subcellular localization has not been clearly defined for most pathogens. Salmonella enterica serovar Typhimurium is not only a leading cause of human gastroenteritis in high-income countries but is also one of the main causes of invasive non-typhoidal salmonellosis (iNTS) in middle- and low-income countries. The incidence of non-typhoidal salmonellosis is increasing worldwide, causing millions of infections and deaths among humans each year. Regrettably, antimicrobial resistance to a broad spectrum of antibiotics is common among non-Typhi Salmonella strains. Therefore, the development of vaccines targeting this leading invasive pathogen is warranted. In the present study we have identified the OM protein profile of the virulent S. Typhimurium strain SL1344 by means of sarkosyl extraction.Salmonella enterica serovar Typhimurium causes food-borne gastroenteritis around the world, but is also responsible for a more serious manifestation of the disease through a form of invasive illness, invasive non-typhoidal Salmonella (iNTS) disease, which is considered a major public health problem in low- and middle-income countries. Even though some studies have been carried out in order to characterize the outer membrane subproteome of this human pathogen, as far as we know, this is the first report in which the most indicated methodology has been used in order to extract the outer membrane proteins and to check the presence of the proteins in the SL1344 genome; indeed all the previous studies were carried out before the genome sequence was available in 2012. Outer membrane proteins are key elements for the interaction of Gram-negative bacteria with their environment – including the host – and have fundamental roles in both infection and resistance processes. Therefore, a detailed knowledge of the outer membrane composition will certainly play a key role in providing new targets to fight this pathogen in further studies.

Objectives This study was focused on analysing the heterogeneity of mutations occurring in the regulators of efflux-mediated MDR in Salmonella Typhimurium. Moreover, the impact of such mutations on impairing the transcription of ramA, acrB, tolC and acrF was also assessed as was the impact on the resistance or decreased susceptibility phenotype. Methods Strains were selected in vitro under increasing ciprofloxacin concentrations. Etest and broth microdilution tests were used to determine the MICs of several unrelated compounds. Screening of mutations in the quinolone target genes and MDR regulators was performed. RT-PCR analysis was used to detect the levels of expression of acrB, tolC, ompF, acrF, emrB, acrR, ramA, soxS and marA. Results All mutant strains showed increased MICs of most of the antimicrobials tested, with the exception of kanamycin. Mutations in the quinolone target genes did not occur in all the mutants, which all harboured mutations in the ramRA regulatory region. All the mutants overexpressed ramA, tolC and acrB (only tested in 60-wt derivatives), whereas differential results were seen for the remaining genes. Conclusions Mutations in the ramRA region related to resistance and/or decreased susceptibility to antimicrobials predominate in Salmonella. There is heterogeneity in the types of mutations, with deletions affecting RamR-binding sites having a greater impact on ramA expression and the MDR phenotype.

Objectives The relationship between quinolone resistance acquisition and invasion impairment has been studied in some Salmonella enterica serovars. However, little information has been reported regarding the invasive human-restricted pathogen Salmonella Typhi. The aim of this study was to investigate the molecular mechanisms of quinolone resistance acquisition and its impact on virulence in this serovar. Methods Two antibiotic-resistant mutants (Ty_c1 and Ty_c2) were generated from a Salmonella Typhi clinical isolate (Ty_wt). The three strains were compared in terms of antimicrobial susceptibility, molecular mechanisms of resistance, gene expression of virulence-related factors, ability to invade eukaryotic cells (human epithelial cells and macrophages) and cytokine production. Results Multidrug resistance in Ty_c2 was attributed to AcrAB/TolC overproduction, decreased OmpF (both mediated by the mar regulon) and decreased OmpC. The two mutants showed a gradually reduced expression of virulence-related genes (invA, hilA, hilD, fliC and fimA), correlating with decreased motility, reduced infection of HeLa cells and impaired uptake by and intracellular survival in human macrophages. Moreover, Ty_c2 also showed reduced tviA expression. Additionally, we revealed a significant reduction in TNF-α and IL-1β production and decreased NF-κB activation. Conclusions In this study, we provide an in-depth characterization of the molecular mechanisms of antibiotic resistance in the Salmonella Typhi serovar and evidence that acquisition of antimicrobial resistance is concomitantly detected with a loss of virulence (epithelial cell invasion, macrophage phagocytosis and cytokine production). We suggest that the low prevalence of clinical isolates of Salmonella Typhi highly resistant to ciprofloxacin is due to poor immunogenicity and impaired dissemination ability of these isolates.

Yersinia enterocolitica are zoonotic bacteria, capable of being transmitted from animals to humans via ingestion of contaminated products. This pathogen causes mainly enterocolitis characterized by fever, watery diarrhea, and abdominal pain. Y. enterocolitica strains can be classified into biotypes or serotypes based on biochemical tests and the somatic O antigen, with six biotypes (1A, 1B and 2–5) and more than 57 serotypes. In both humans and animals, bioserotype 4/O:3 is the most prevalent type isolated worldwide. Currently, most Y. enterocolitica isolates are susceptible to aminoglycosides, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, ciprofloxacin, and third-generation cephalosporins in vitro. The last two antibiotics, in combination with gentamicin, represent the treatment of choice for enteritis and systemic infections, respectively. However, resistance to several of these compounds has been reported and the mechanisms to the most representative families (β-lactams, aminoglycosides, and quinolones) are described in this chapter.

Received 11 November 2013; returned 12 December 2013; revised 15 February 2014; accepted 5 March 2014Objectives: To investigate the potential relationship between quinolone resistance and biofilm production in acollection of Salmonella enterica clinical isolates and inS. enterica serovar Typhimurium serial mutants withincreasing resistance to ciprofloxacin.Methods:Nalidixicacidsusceptibilityandbiofilmformationwereassessedinacollectionof122S.entericaclinicalisolates. An in vitro quinolone-resistant mutant, 59-64, was obtained from a biofilm-producing and quinolone-susceptible clinical isolate, 59-wt, in a multistep selection process after increasing ciprofloxacin concentrations.The quinolone resistance mechanisms [target gene and multidrug resistance (MDR) regulatory mutations, MICsof several antibiotics, cell envelope protein analysis, real-time PCR and ciprofloxacin accumulation] were charac-terized for mutant strains. In addition, analysis of fitness, biofilm formation, rdar morphotype and expression ofbiofilm-related genes by real-time PCR were also determined.Results: Nalidixic acid-susceptible S. enterica strains were more prevalent in producing biofilm than the resistantcounterparts. Strain 59-64 acquired five target gene mutations and showed an MDR phenotype. AcrAB andacrF overexpression were ruled out, whereas TolC did show increased expression in 59-64, which, in addition,accumulated less ciprofloxacin. Consistently, increased ramA expression was seen in 59-64 and attributed to amutation within its promoter. Reduced biofilm production related to diminishedcsgB expression as well asreduced fitness was seen for 59-64, which was unable to form the rdar morphotype.Conclusions: Quinolone resistance acquisition may be associated with decreased production of biofilm due tolower csgB expression. Efflux, biofilm production and fitness seem to be interrelated.Keywords: Salmonella Typhimurium, rdar morphotype, csgB, efflux, ramA