Insights into the Evolution of IncR Plasmids Found in the Southern European Clone of the Monophasic Variant of Salmonella enterica Serovar Typhimurium.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium which has emerged as a world-wide distributed pathogen in the last decades. Several clones have been identified within this variant, the European clone, the Spanish clone, the Southern European clone and the U.S./American clone. The present study focused on isolates of the Southern European clone that were obtained from clinical samples at Spanish hospitals. The selected isolates were multidrug resistant, with most resistance genes residing on IncR plasmids that also carried virulence genes. These plasmids had a mosaic structure, comprising a highly reduced IncR backbone, which has acquired a large amount of exogenous DNA mostly derived from pSLT and IncI1-I(alfa) plasmids. Although composed of approximately the same elements, the investigated plasmids displayed a high diversity, consistent with active evolution driven by a wealth of mobile genetic elements. They comprise multiple intact or truncated insertion sequences, transposons, pseudo-compound transposons and integrons. Particularly relevant was the role of IS26 (with six to nine copies per plasmid) in generating insertions, deletions and inversions, with many of the rearrangements uncovered by tracking the patterns of eight bp target site duplications. Most of the resistance genes detected in the analyzed isolates have been previously associated with the Southern European clone. However, erm(B), lnu(G) and blaTEM-1B are novel, with the last two carried by a second resistance plasmid found in one of the IncR-positive isolates. Thus, evolution of resistance in the Southern European clone is not only mediated by diversification of the IncR plasmids, but also through acquisition of additional plasmids. All isolates investigated in the present study have the large deletion affecting the fljBA region previously found to justify the monophasic phenotype in the Southern European and U.S./American clones. An SNP-based phylogenetic analysis revealed the close relationship amongst our isolates, and support that those sharing the large fljBA deletion could be more heterogeneous than previously anticipated.

Study of the intestinal microbiota composition and the effect of treatment with intensive chemotherapy in patients recovered from acute leukemia.

A dataset comprising metagenomes of outpatients (n = 28) with acute leukemia (AL) and healthy controls (n = 14) was analysed to investigate the associations between gut microbiota composition and metabolic activity and AL. According to the results obtained, no significant differences in the microbial diversity between AL outpatients and healthy controls were found. However, significant differences in the abundance of specific microbial clades of healthy controls and AL outpatients were found. We found some differences at taxa level. The relative abundance of Enterobacteriaceae, Prevotellaceae and Rikenellaceae was increased in AL outpatients, while Bacteirodaceae, Bifidobacteriaceae and Lachnospiraceae was decreased. Interestingly, the abundances of several taxa including Bacteroides and Faecalibacterium species showed variations based on recovery time from the last cycle of chemotherapy. Functional annotation of metagenome-assembled genomes (MAGs) revealed the presence of functional domains corresponding to therapeutic enzymes including L-asparaginase in a wide range of genera including Prevotella, Ruminococcus, Faecalibacterium, Alistipes, Akkermansia. Metabolic network modelling revealed potential symbiotic relationships between Veillonella parvula and Levyella massiliensis and several species found in the microbiota of AL outpatients. These results may contribute to develop strategies for the recovery of microbiota composition profiles in the treatment of patients with AL.

Usefulness of inclusion of ertapenem and temocillin screening breakpoints in the EUCAST rapid antimicrobial susceptibility testing (RAST) for rapid detection of OXA-48-producing Klebsiella pneumoniae directly from positive blood cultures.

OBJECTIVES: The aims of this study were: (i) to assess the ability of the meropenem screening breakpoint as part of the screening rapid antimicrobial susceptibility testing (sRAST) of EUCAST for the detection of OXA-48 carbapenemase-producing Klebsiella pneumoniae directly from positive blood cultures (BCs); and (ii) to evaluate the inclusion of ertapenem and temocillin discs into the sRAST to enhance the detection of OXA-48-producing isolates. METHODS: BC bottles were spiked with a total of 117 K. pneumoniae isolates, including 77 previously characterized OXA-48 producers and 40 non-OXA-48 producers. Disc diffusion assays were directly performed from positive BCs with meropenem (10 µg), ertapenem (10 µg) and temocillin (30 µg) discs, and inhibition zones were manually measured after 4, 6 and 8 h of incubation. The screening cut-off values of sRAST were applied to evaluate their capability in detecting OXA-48-producing isolates. Receiver operating characteristic curves were constructed to illustrate the performance efficacy of the disc diffusion assays to detect OXA-48 producers. RESULTS: The meropenem cut-off values of sRAST only detected 90.91% of the OXA-48-producing isolates after 6 and 8 h of incubation. With the proposed cut-off points for ertapenem [<19 mm (4/6 h) and <20 mm (8 h)] and temocillin [<10 mm (4 h) and <11 mm (6/8 h)], all OXA-48-positive isolates were detected without any false-positive results at any reading time. CONCLUSIONS: In healthcare settings with a high prevalence of OXA-48 producers, the inclusion of ertapenem and temocillin discs in the sRAST procedure may improve the detection of OXA-48-producing K. pneumoniae isolates directly from positive BCs, providing reliable results after only a 4 h incubation period.

A highly-safe live auxotrophic vaccine protecting against disease caused by non-typhoidal Salmonella Typhimurium in mice.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive infections in populations at risk. There is at present an unmet need for preventive vaccines. METHODS: IRTA GN-3728 genome was sequenced by Illumina and d-glutamate and d-glutamate/d-alanine knockout-auxotrophs were constructed. They were characterized using electron microscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated. RESULTS: The d-glutamate auxotroph, IRTA ΔmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA ΔmurI Δalr ΔdadX double auxotroph, constructed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effective against systemic disease. In the intestinal model, it proved to be safe, yielding a low-degree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homologous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection. CONCLUSION: This study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans.

Incidence and Genomic Background of Antibiotic Resistance in Food-Borne and Clinical Isolates of Salmonella enterica Serovar Derby from Spain.

Salmonella enterica serovar Derby (S. Derby) ranks fifth among nontyphoidal Salmonella serovars causing human infections in the European Union. S. Derby isolates (36) collected between 2006 and 2018 in a Spanish region (Asturias) from human clinical samples (20) as well as from pig carcasses, pork- or pork and beef-derived products, or wild boar (16) were phenotypically characterized with regard to resistance, and 22 (12 derived from humans and 10 from food-related samples) were also subjected to whole genome sequence analysis. The sequenced isolates belonged to ST40, a common S. Derby sequence type, and were positive for SPI-23, a Salmonella pathogenicity island involved in adherence and invasion of the porcine jejune enterocytes. Isolates were either susceptible (30.6%), or resistant to one or more of the 19 antibiotics tested for (69.4%). Resistances to tetracycline [tet(A), tet(B) and tet(C)], streptomycin (aadA2), sulfonamides (sul1), nalidixic acid [gyrA (Asp87 to Asn)] and ampicillin (blaTEM-1-like) were detected, with frequencies ranging from 8.3% to 66.7%, and were higher in clinical than in food-borne isolates. The fosA7.3 gene was present in all sequenced isolates. The most common phenotype was that conferred by the tet(A), aadA2 and sul1 genes, located within identical or closely related variants of Salmonella Genomic Island 1 (SGI1), where mercury resistance genes were also present. Diverse IncI1-I(α) plasmids belonging to distinct STs provided antibiotic [blaTEM-1, tet(A) and/or tet(B)] and heavy metal resistance genes (copper and silver), while small pSC101-like plasmids carried tet(C). Regardless of their location, most resistance genes were associated with genetic elements involved in DNA mobility, including a class one integron, multiple insertion sequences and several intact or truncated transposons. By phylogenetic analysis, the isolates were distributed into two distinct clades, both including food-borne and clinical isolates. One of these clades included all SGI1-like positive isolates, which were found in both kinds of samples throughout the entire period of study. Although the frequency of S. Derby in Asturias was very low (0.5% and 3.1% of the total clinical and food isolates of S. enterica recovered along the period of study), it still represents a burden to human health linked to transmission across the food chain. The information generated in the present study can support further epidemiological surveillance aimed to control this zoonotic pathogen.

Spread of blaCTX-M-9 and Other Clinically Relevant Resistance Genes, Such as mcr-9 and qnrA1, Driven by IncHI2-ST1 Plasmids in Clinical Isolates of Monophasic Salmonella enterica Serovar Typhimurium ST34.

The monophasic 4,[5],12:i:-variant of Salmonella enterica serovar Typhimurium with sequence type ST34 has become one of the most prevalent non-typhoidal salmonellae worldwide. In the present study, we thoroughly characterized seven isolates of this variant detected in a Spanish hospital and selected based on cefotaxime resistance and cefoxitin susceptibility, mediated by blaCTX-M-9. For this, conventional microbiological techniques, together with whole genome sequencing performed with the Illumina platform, were applied. All selected isolates carried the resistance region RR or variants therein, and most also contained the SGI-4 genomic island. These chromosomal elements, typically associated with monophasic S. Typhimurium ST34, confer resistance to traditional antibiotics (ampicillin, streptomycin, sulfonamides, and tetracycline) and tolerance to heavy metals (mercury, silver, and copper). In addition, each isolate carried a large IncHI2-ST1 conjugative plasmid containing additional or redundant resistance genes. All harbored the blaCTX-M-9 gene responsible for cefotaxime resistance, whereas the qnrA1 gene mediating fluoroquinolone resistance was detected in two of the plasmids. These genes were embedded in ISCR1-bearing complex class 1 integrons, specifically In60-like and In36-like. The mcr-9 gene was present in all but one of the IncHI2-ST1 plasmids found in the analyzed isolates, which were nevertheless susceptible to colistin. Most of the resistance genes of plasmid origin clustered within a highly complex and variable region. The observed diversity results in a wide range of resistance phenotypes, enabling bacterial adaptation to selective pressure posed by the use of antimicrobials.

High-risk international clones ST66, ST171 and ST78 of Enterobacter cloacae complex causing blood stream infections in Spain and carrying blaOXA-48 with or without mcr-9.

BACKGROUND: In the last years, Enterobacter cloacae complex has become an important threat associated with nosocomial infections (including bacteraemia). These bacteria have the ability to acquire mobile genetic elements with antimicrobial resistance genes, reducing the number of therapies available for treatment of the infections they cause. Multidrug resistant isolates of the E. cloacae complex have been causing blood stream infections in a hospital in northern Spain. The aim of this study was to report the spread of E. cloacae complex isolates carrying blaOXA-48 with or without mcr-9 which were involved in blood stream infections, in a Spanish hospital. METHODS: All Enterobacter spp. isolates recovered from blood cultures of patients admitted to a tertiary Spanish hospital, over a five-year period were recovered. Of those, OXA-48-producing isolates were selected for further analysis (19 E. xiangfangensis isolates and a single E. hoffmannii). Bacterial identification, antimicrobial susceptibility, DNA sequencing, molecular typing, resistome analysis and plasmid characterization was performed. RESULTS: 20 isolates were positive for blaOXA-48, harbored by IncL/M plasmids. They belonged to the international high-risk clones ST66, ST171 and ST78. They produced the extended-spectrum ß-lactamases CTX-M-15 and/or CTX-M-9 and 40 % of them (n = 8) also carried the mcr-9 gene, located on IncHI2 plasmids. However, they were susceptible to colistin. CONCLUSION: The presence of blaOXA-48, together with at least one blaCTX-M gene in our multidrug resistant high-risk E. cloacae complex clones is worrisome. Also, the additional presence of mcr-9 in some of them is of concern as it could potentially be transferred into other hosts or acquire mutations that might led to emerging colistin resistance. Surveillance systems are essential to detect these difficult-to-treat bacteria which, apart from causing live-threatening infections, can spread important resistance threats.

Biochemical Diversity, Pathogenicity and Phylogenetic Analysis of Pseudomonas viridiflava from Bean and Weeds in Northern Spain.

Pseudomonas viridiflava was originally reported as a bean pathogen, and subsequently as a wide-host range pathogen affecting numerous plants species. In addition, several authors have reported the epiphytic presence of this bacterium in "non-host plants", which may act as reservoir of P. viridiflava and source of inoculum for crops. A new biotype of this bacterium, showing an atypical LOPAT profile, was found in Asturias, a Northern region of Spain, causing significant damage in beans, kiwifruit, lettuce, and Hebe. In order to investigate the involvement of weeds in bean disease, samples were collected from beans and weeds growing in the same fields. A total of 48 isolates of P. viridiflava were obtained, 39 from weeds and 9 from beans. 48% and 52% of them showed typical (L- O- P+ A- T+) and atypical (L+ O- P v A- T+) LOPAT profiles, and they displayed high biochemical diversity. Regarding virulence factors, the T-PAI and S-PAI pathogenicity islands were found in 29% and 70.8% of the isolates, 81.2% displayed pectinolytic activity on potato slices, and 59% of the weed isolates produced symptoms after inoculation on bean pods. A phylogenetic tree based on concatenated rpoD, gyrB, and gltA sequences separated the strains carrying S-PAI and T-PAI into different clusters, both containing isolates from beans and weeds, and pathogenic as well as non-pathogenic strains. Closely related strains were found in the two hosts, and more than half of the weed isolates proved to be pathogenic in beans. This is consistent with the role of weeds as a reservoir and source of inoculum for bean infection. Detection of P. viridiflava in weeds throughout the year further supports these roles.

Genomic Analysis of Two MDR Isolates of Salmonella enterica Serovar Infantis from a Spanish Hospital Bearing the blaCTX-M-65 Gene with or without fosA3 in pESI-like Plasmids.

Salmonella enterica serovar Infantis (S. Infantis) is a broiler-associated pathogen which ranks in the fourth position as a cause of human salmonellosis in the European Union. Here, we report a comparative genomic analysis of two clinical S. Infantis isolates recovered in Spain from children who just returned from Peru. The isolates were selected on the basis of resistance to cefotaxime, one of the antibiotics of choice for treatment of S. enterica infections. Antimicrobial susceptibility testing demonstrated that they were resistant to eight classes of antimicrobial agents: penicillins, cephalosporins, phenicols, aminoglycosides, tetracyclines, inhibitors of folate synthesis, (fluoro)quinolones and nitrofurans, and one of them was also resistant to fosfomycin. As shown by whole-genome sequence analysis, each isolate carried a pESI-like megaplasmid of ca. 300 kb harboring multiple resistance genes [blaCTX-M-65, aph(4)-Ia, aac(3)-IVa, aph(3')-Ia, floR, dfrA14, sul1, tet(A), aadA1 ± fosA3], as well as genes for resistance to heavy metals and disinfectants (mer, ars and qacEΔ1). These genes were distributed in two complex regions, separated by DNA belonging to the plasmid backbone, and associated with a wealth of transposable elements. The two isolates had a D87Y amino acid substitution in the GyrA protein, and truncated variants of the nitroreductase genes nfsA and nsfB, accounting for chromosomally encoded resistances to nalidixic acid and nitrofurantoin, respectively. The two S. Infantis isolates were assigned to sequence type ST32 by in silico multilocus sequence typing (MLST). Phylogenetic analysis revealed that they were closely related, differing only by 12 SNPs, although they were recovered from different children two years apart. They were also genetically similar to blaCTX-M-65-positive ± fosA3 isolates obtained from humans and along the poultry production chain in the USA, South America, as well as from humans in several European countries, usually associated with a travel history to America. However, this is the first time that the S. Infantis blaCTX-M-65 ± fosA3 MDR clone has been reported in Spain.

Genomic Analysis of Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 From Spanish Hospitals.

Salmonella enterica serovar Kentucky (S. Kentucky) with sequence type (ST) 198 and highly resistant to ciprofloxacin (ST198-Cip R ) has emerged as a global MDR clone, posing a threat to public health. In the present study, whole genome sequencing (WGS) was applied to characterize all Cip R S. Kentucky detected in five Spanish hospitals during 2009-2018. All Cip R isolates (n = 13) were ST198 and carried point mutations in the quinolone resistance-determining regions (QRDRs) of both gyrA (resulting in Ser83Phe and Asp87Gly, Asp87Asn, or Asp87Tyr substitutions in GyrA) and parC (with Thr57Ser and Ser80Ile substitutions in ParC). Resistances to other antibiotics (ampicillin, chloramphenicol, gentamicin, streptomycin, sulfonamides, and tetracycline), mediated by the bla TEM- 1 B , catA1, aacA5, aadA7, strA, strB, sul1, and tet(A) genes, and arranged in different combinations, were also observed. Analysis of the genetic environment of the latter resistance genes revealed the presence of multiple variants of SGI1 (Salmonella genomic island 1)-K and SGI1-P, where all these resistance genes except catA1 were placed. IS26 elements, found at multiple locations within the SGI1 variants, have probably played a crucial role in their generation. Despite the wide diversity of SGI1-K- and SGI1-P-like structures, phylogenetic analysis revealed a close relationship between isolates from different hospitals, which were separated by a minimum of two and a maximum of 160 single nucleotide polymorphisms. Considering that S. enterica isolates resistant to fluoroquinolones belong to the high priority list of antibiotic-resistant bacteria compiled by the World Health Organization, continuous surveillance of the S. Kentucky ST198-CIP R clone is required.

Genomic analysis and phylogenetic position of the complex IncC plasmid found in the Spanish monophasic clone of Salmonella enterica serovar Typhimurium.

pUO-STmRV1 is an IncC plasmid discovered in the Spanish clone of the emergent monophasic variant of Salmonella enterica serovar Typhimurium, which has probably contributed to its epidemiological success. The sequence of the entire plasmid determined herein revealed a largely degenerated backbone with accessory DNA incorporated at four different locations. The acquired DNA constitutes more than two-thirds of the pUO-STmRV1 genome and originates from plasmids of different incompatibility groups, including IncF (such as R100 and pSLT, the virulence plasmid specific of S. Typhimurium), IncN and IncI, from the integrative element GIsul2, or from yet unknown sources. In addition to pSLT virulence genes, the plasmid carries genes conferring resistance to widely-used antibiotics and heavy metals, together with a wealth of genetic elements involved in DNA mobility. The latter comprise class 1 integrons, transposons, pseudo-transposons, and insertion sequences, strikingly with 14 copies of IS26, which could have played a crucial role in the assembly of the complex plasmid. Typing of pUO-STmRV1 revealed backbone features characteristically associated with type 1 and type 2 IncC plasmids and could therefore be regarded as a hybrid plasmid. However, a rooted phylogenetic tree based on core genes indicates that it rather belongs to an ancient lineage which diverged at an early stage from the branch leading to most extant IncC plasmids detected so far. pUO-STmRV1 may have evolved at a time when uncontrolled use of antibiotics and biocides favored the accumulation of multiple resistance genes within an IncC backbone. The resulting plasmid thus allowed the Spanish clone to withstand a wide variety of adverse conditions, while simultaneously promoting its own propagation through vertical transmission.

Colistin Resistance in Monophasic Isolates of Salmonella enterica ST34 Collected From Meat-Derived Products in Spain, With or Without CMY-2 Co-production.

Colistin is a last-resort antibiotic in fighting severe infections caused by multidrug resistant Gram negative pathogens in hospitals. Zoonotic bacteria acquire colistin resistance in animal reservoirs and mediate its spread along the food chain. This is the case of non-typhoid serovars of Salmonella enterica. Colistin-resistant S. enterica in foods represents a threat to human health. Here, we assessed the prevalence of colistin-resistance in food-borne isolates of S. enterica (2014-2019; Asturias, Spain), and established the genetic basis and transferability of this resistance. Five out of 231 isolates tested (2.2%) were resistant to colistin. Four of them, belonging to the European monophasic ST34 clone of S. Typhimurium, were characterized in the present study. They were collected from pork or pork and beef meat-derived products, either in 2015 (three isolates) or 2019 (one isolate). Molecular typing with XbaI-PFGE and plasmid profiling revealed distinct patterns for each isolate, even though two of the 2015 isolates derived from the same sample. The MICs of colistin ranged from 8 to 16 mg/L. All isolates carried the mcr-1.1 gene located on conjugative plasmids of the incompatibility groups IncX4 (2015 isolates) or IncHI2 (2019 isolate). Apart from colistin resistance, the four isolates carried chromosomal genes conferring resistance to ampicillin, streptomycin, sulfonamides and tetracycline [bla TEM-1, strA-strB, sul2, and tet(B)] and heavy metals, including copper and silver (silESRCFBAGP and pcoGE1ABCDRSE2), arsenic (arsRSD2A2BCA1D1) ± mercury (merEDACPTR), which are characteristically associated with the European ST34 monophasic clone. The 2019 isolate was also resistant to other antibiotics, comprising third generation cephalosporins and cephamycins. The latter phenotype was conferred by the bla CMY-2 gene located on an IncI1-I(α)-ST2 plasmid. Results in the present study identified meat-derived products as a reservoir of a highly successful clone harboring transferable plasmids which confer resistance to colistin and other clinically important antibiotics. An important reduction in the number of food-borne S. enterica detected during the period of the study, together with the low frequency of colistin resistance, underlines the success of One Health initiatives, such as those implemented at the UE, to control zoonotic bacteria along the food chain and to halt the spread of antimicrobial resistance.

Extensively drug-resistant Acinetobacter baumannii carrying blaOXA-23-like and armA in a hospital after an intervention in the intensive care unit which ended a long-standing endemicity.

The aim of the study was to evaluate for a long time the effectiveness of an intervention designed to reduce carbapenem-resistant Acinetobacter baumannii (CRAB) and its impact on colistin usage in the ICU of a tertiary hospital in Spain. The rate of carbapenem resistance declined drastically during the period of study (2015 to 2018), from 93.57 to 74.65%, especially in the ICU. A significant decrease in colistin usage, from 1.16 to 0.39 DOTs, was observed. Forty-nine CRAB isolates recovered nearly 1 year after starting the intervention were characterized. Most of them were recovered from patients admitted in wards other than ICU and were extensively drug-resistant, carried blaOXA-23-like and armA, and belonged to ST218. Implementation of control measures is crucial to CRAB control in ICUs but must be extended to all wards in order to eradicate CRAB from hospitals.

Molecular Characterization of Salmonella enterica Serovar Enteritidis, Genetic Basis of Antimicrobial Drug Resistance and Plasmid Diversity in Ampicillin-Resistant Isolates.

Salmonella enterica serovar Enteritidis is the most common cause of human salmonellosis worldwide. In this study, all clinical isolates of Salmonella Enteritidis recovered between January 2008 and June 2014 in a Spanish region (491) were screened for antimicrobial drug resistance and the phage type (PT) was determined for a significant number (265). PT1, PT14b, PT56, PT6, PT4, and PT8 were the predominant PTs, accounting together for 82% of the isolates. A total of 38.3% of the isolates were susceptible to all antimicrobials tested, 46.4% and 6.1% isolates were resistant to nalidixic acid and ampicillin, respectively, and single isolates were resistant to two (ampicillin and nalidixic acid) or six (ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim) agents. Nalidixic acid resistance was statistically associated with PT1 and PT14b (p < 0.05, 95% CI), and ampicillin resistance with PT6/PT6a (p < 0.05, 95% CI). All ampicillin-resistant isolates (30) carried a plasmid-encoded blaTEM-1. All except one harbored the virulence plasmid specific of Salmonella Enteritidis (IncFIIA + IncFIB; 28 isolates) or a blaTEM-1-positive variant herein (IncFIIA + IncFIB; 1 isolate). Five additional blaTEM-1 plasmids, of the ColE1, IncX, IncF, and IncI incompatibility groups, were identified. The IncI plasmid, found in the single multidrug-resistant isolate, carried the strAB and sul2 genes together with genes of the virulence plasmid, including the spv operon. The obtained results highlight the high diversity of blaTEM-1 plasmids conferring ampicillin resistance in Salmonella Enteritidis, and support clonal expansion as the main cause of nalidixic acid resistance in this serovar.

Human Pasteurella multocida Infection with Likely Zoonotic Transmission from a Pet Dog, Spain.

We report a case of urinary tract infection caused by an unusual genotype (sequence type 211) of Pasteurella multocida associated with human infection. Molecular genetic analysis of P. multocida isolates obtained from the human patient and his pet strongly suggests a zoonotic transmission of this bacterium.

Resistance to Carbapenems in Non-Typhoidal Salmonella enterica Serovars from Humans, Animals and Food.

Non-typhoidal serovars of Salmonella enterica (NTS) are a leading cause of food-borne disease in animals and humans worldwide. Like other zoonotic bacteria, NTS have the potential to act as reservoirs and vehicles for the transmission of antimicrobial drug resistance in different settings. Of particular concern is the resistance to critical "last resort" antimicrobials, such as carbapenems. In contrast to other Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli, and Enterobacter, which are major nosocomial pathogens affecting debilitated and immunocompromised patients), carbapenem resistance is still very rare in NTS. Nevertheless, it has already been detected in isolates recovered from humans, companion animals, livestock, wild animals, and food. Five carbapenemases with major clinical importance-namely KPC (Klebsiella pneumoniae carbapenemase) (class A), IMP (imipenemase), NDM (New Delhi metallo-ß-lactamase), VIM (Verona integron-encoded metallo-ß-lactamase) (class B), and OXA-48 (oxacillinase, class D)-have been reported in NTS. Carbapenem resistance due to the production of extended spectrum- or AmpC ß-lactamases combined with porin loss has also been detected in NTS. Horizontal gene transfer of carbapenemase-encoding genes (which are frequently located on self-transferable plasmids), together with co- and cross-selective adaptations, could have been involved in the development of carbapenem resistance by NTS. Once acquired by a zoonotic bacterium, resistance can be transmitted from humans to animals and from animals to humans through the food chain. Continuous surveillance of resistance to these "last resort" antibiotics is required to establish possible links between reservoirs and to limit the bidirectional transfer of the encoding genes between S. enterica and other commensal or pathogenic bacteria.

Evaluation of Sepsis Flow Chip for identification of Gram-negative bacilli and detection of antimicrobial resistance genes directly from positive blood cultures.

Blood stream infections are serious conditions associated with high morbi-mortality. In this study, the new Sepsis Flow Chip (SFC) assay for identification of Gram-negative bacteria and their antimicrobial resistance genes was evaluated in positive blood cultures (BCs). SFC is a microarray with a broad panel comprising the most frequent causative agents of sepsis and antimicrobial resistance genes associated with them. A total of 100 prospective BCs, positive for Gram-negative bacilli, were assessed in the routine of the clinical microbiology laboratory and also applying the SFC assay. Moreover, 19 BCs spiked with well-characterized enterobacterial isolates, harboring antimicrobial resistance genes, were analyzed by the latter. Among the monomicrobial BCs (90), the concordance between SFC identification and the reference method was 94.4%; however, it achieved 100% when SFC was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after 4-h incubation. Regarding polymicrobial BCs (10), 15 out of the 22 bacteria present (68.2%) were correctly identified, including all contained in 50% of the cultures. With regard to antimicrobial resistance genes, 98.8%, 98.9%, and 99% concordance was obtained for blaCTX-M, blaOXA-48, and blaVIM, respectively, in comparison with polymerase chain reaction amplification. SFC assay gives results in only 4 h and showed a high concordance rate with the reference method. Although further evaluation studies are necessary, SFC assay implementation, together with antimicrobial stewardship programs, could contribute to improve the therapeutic approaches and to reduce the morbi-mortality, length of hospital stay, and healthcare-associated costs in patients with sepsis.

Salmonella enterica serovars Typhimurium and Enteritidis causing mixed infections in febrile children in Mozambique.

BACKGROUND AND PURPOSE: Invasive nontyphoidal salmonellosis, mostly caused by serovars Typhimurium and Enteritidis of Salmonella enterica, has emerged as a major public health problem in sub-Saharan Africa. The aim of this study was the clinical and microbiological characterization of nontyphoidal salmonellosis episodes affecting febrile children in Mozambique. PATIENTS AND METHODS: The clinical records of the patients were evaluated, and S. enterica isolates were characterized with regard to serovar, phage type, antimicrobial resistance (phenotype/responsible genes), plasmid content, pulsed-field gel electrophoresis, and multilocus sequence typing. RESULTS: Fifteen S. Typhimurium and 21 S. Enteritidis isolates were recovered from blood samples of 25 children, the majority with underlying risk factors. With regard to phage typing, most isolates were either untypeable or reacted but did not conform, revealing that a number of previously unrecognized patterns are circulating in Mozambique. Most isolates were multidrug-resistant, with nearly all of the responsible genes located on derivatives of serovar-specific virulence plasmids. ST313 and ST11 were the predominant sequence types associated with S. Typhimurium and S. Enteritidis, respectively, and the uncommon ST1479 was also detected in S. Enteritidis. A distinct XbaI fragment of ~350 kb was associated with pulsed-field gel electrophoresis patterns of multidrug-resistant isolates of S. Enteritidis. Nearly half of the children were coinfected with both serovars, a fact expected to aggravate the disease and hamper the treatment. However, particularly poor outcomes were not observed for the coinfected patients. CONCLUSION: Mixed Salmonella infections could frequently occur in febrile children in Mozambique. Additional studies are required to determine their actual impact and consequences, not only in this country, but also in other African countries.

Whole genome sequencing, molecular typing and in vivo virulence of OXA-48-producing Escherichia coli isolates including ST131 H30-Rx, H22 and H41 subclones.

Carbapenem-resistant Enterobacteriaceae, including the increasingly reported OXA-48 Escherichia coli producers, are an emerging public health threat worldwide. Due to their alarming detection in our healthcare setting and their possible presence in the community, seven OXA-48-producing, extraintestinal pathogenic E. coli were analysed by whole genome sequencing as well as conventional tools, and tested for in vivo virulence. As a result, five E. coli OXA-48-producing subclones were detected (O25:H4-ST131/PST43-fimH30-virotype E; O25:H4-ST131/PST9-fimH22-virotype D5, O16:H5-ST131/PST506-fimH41; O25:H5-ST83/PST207 and O9:H25-ST58/PST24). Four ST131 and one ST83 isolates satisfied the ExPEC status, and all except the O16:H5 ST131 isolate were UPEC. All isolates exhibited local inflammatory response with extensive subcutaneous necrosis but low lethality when tested in a mouse sepsis model. The bla OXA-48 gene was located in MOBP131/IncL plasmids (four isolates) or within the chromosome (three ST131 H30-Rx isolates), carried by Tn1999-like elements. All, except the ST83 isolate, were multidrug-resistant, with additional plasmids acting as vehicles for the spread of various resistance genes. This is the first study to analyse the whole genome sequences of bla OXA-48-positive ST131, ST58 and ST83 E. coli isolates in conjunction with experimental data, and to evaluate the in vivo virulence of bla OXA-48 isolates, which pose an important challenge to patient management.