KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: blaKPC-80, blaKPC-81, blaKPC-96 and blaKPC-97.

Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of blaKPC-2 resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant blaKPC genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for blaKPC-80, blaKPC-96, and blaKPC-97 by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including blaTEM-1, blaOXA-18 and blaOXA-1, were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of blaKPC-2 and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, blaKPC-2. The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

National action plans on antimicrobial resistance in Latin America: an analysis via a governance framework.

In 2015, the World Health Assembly adopted a global action plan (GAP) on antimicrobial resistance (AMR). Member states were encouraged to develop their own national action plans (NAPs) in alignment with the GAP. To-date, in systematic assessments of NAPs, the Latin American specific context has not been previously analysed. Here we examined 11 Latin American NAPs published between 2015 and 2021 using content analysis. We focused on two approaches: (1) alignment between the strategic objectives and actions defined in the GAP, and those outlined in the NAPs via a content indicator; and (2) assessment of the NAPs via a governance framework covering 'policy design', 'implementation tools' and 'monitoring and evaluation' areas. We observed a high alignment with the strategic objectives of the GAP; however, the opposite was observed for the corresponding actions. Our results showed that the governance aspects contained within coordination and participation domains were addressed by every Latin American NAP, whereas monitoring and assessment areas, as well as incorporating the environment, would need more attention in subsequent NAPs. Given that AMR is a global health threat and collective efforts across regions are necessary to combat it, our findings can benefit member states by highlighting how to strengthen the AMR strategies in Latin America, while also supporting global policy formulation.

Co-integrate Col3m bla NDM-1-harboring plasmids in clinical Providencia rettgeri isolates from Argentina.

The first cases of bla NDM in Argentina were detected in three Providencia rettgeri (Pre) recovered from two hospitals in Buenos Aires city in 2013. The isolates were genetically related, but the plasmid profile was different. Here, we characterized the bla NDM-1-harboring plasmids of the first three cases detected in Argentina. Hybrid assembly obtained from short- and long-read sequencing rendered bla NDM-1 in Col3M plasmids of ca. 320 kb (p15268A_320) in isolate PreM15268, 210 kb (p15758B_210) in PreM15758, and 225 kb (p15973A_225) in PreM15973. In addition, PreM15758 harbored a 98-kb circular plasmid (p15758C_98) flanked by a putative recombination site (hin-TnAs2), with 100% nucleotide ID and coverage with p15628A_320. Analysis of PFGE/S1-nuclease gel, Southern hybridization with bla NDM-1 probe, hybrid assembly of short and long reads suggests that pM15758C_98 can integrate by homologous recombination. The three bla NDM-1-plasmids were non-conjugative in vitro. Moreover, tra genes were incomplete, and oriT was not found in the three bla NDM-1-plasmids. In two isolates, blaNDM-1 was embedded in a partially conserved structure flanked by two ISKox2. In addition, all plasmids harbored aph(3')-Ia, aph(3')-VI, and qnrD1 genes and aac(6´)Ib-cr, bla OXA-1, catB3, and arr3 as part of a class 1 integron. Also, p15268A_320 and p15973A_225 harbored bla PER-2. To the best of our knowledge, this is the first report of clinical P. rettgeri harboring blaNDM-1 in an atypical genetic environment and located in unusual chimeric Col3M plasmids. The study and continuous surveillance of these pathogens are crucial to tracking the evolution of these resistant plasmids and finding solutions to tackle their dissemination. IMPORTANCE Infections caused by carbapenem hydrolyzing enzymes like NDM (New Delhi metallo-beta-lactamase) represent a serious problem worldwide because they restrict available treatment options and increase morbidity and mortality, and treatment failure prolongs hospital stays. The first three cases of NDM in Argentina were caused by genetically related P. rettgeri recovered in two hospitals. In this work, we studied the genetic structure of the plasmids encoding bla NDM in those index cases and revealed the enormous plasticity of these genetic elements. In particular, we found a small plasmid that was also found inserted in the larger plasmids by homologous recombination as a co-integrate element. We also found that the bla NDM plasmids were not able to transfer or move to other hosts, suggesting their role as reservoir elements for the acquisition of resistance genes. It is necessary to unravel the dissemination strategies and the evolution of these resistant plasmids to find solutions to tackle their spread.

Novel Metallo-ß-Lactamase blaCVI-1 Isolated from a Chromobaterium violaceum Clinical Strain Resistant to Colistin.

OBJECTIVE: We aimed to describe a colistin (COL)-resistant (R) Chromobacterium violaceum (Cvi) isolate from a septic patient in Argentina expressing a previously unknown gene, blaCVI-1. METHODS: In 2019, a 12 year old child was injured with a thorn in a lagoon. The child was hospitalized due to sepsis and multiple abscesses. Cvi was isolated from skin and soft tissue and tracheal aspirate. The patient was successfully treated with imipenem (IMI) plus amikacin. Antimicrobial susceptibility was assessed by disk diffusion, broth microdilution, and the E-test. Carbapenemase activity was assayed by double-disk synergy and microbiological tests. Resistance, virulence, and additional gene searches were performed by in silico analysis of sequences obtained by whole-genome sequencing (WGS). A maximum likelihood phylogenetic tree was built with public Cvi genomes. RESULTS: R was seen for IMI and COL. Expression of a metallo-ß-lactamase was confirmed. Genome analysis revealed blaCVI-1, a subclass B2 metallo-ß-lactamase with 62.66% ID with CphA from A. hydrophila (WP081086394). R to COL could be attributed to the arnC and arnT genes. Virulence factors required for invasion and toxicity were also found. No plasmids were detected. The phylogeny tree showed two main clades with geographical distinction, and the isolate studied here stands alone in a branch closely related to two clinical isolates from the USA. CONCLUSIONS: This is the second report of infection by Cvi in Argentina. This pathogen carried a new gene, blaCVI-1, a metallo-ß-lactamase that can be detected by routine methods. Prompt suspicion of C. violaceum infection is crucial to treating this rare pathogen rapidly and properly.

Emergence of Hyper-Epidemic Clones of Enterobacterales Clinical Isolates Co-Producing KPC and Metallo-Beta-Lactamases during the COVID-19 Pandemic.

BACKGROUND: The global spread of carbapenemase-producing Enterobacterales has become an epidemiological risk for healthcare systems by limiting available antimicrobial treatments. The COVID-19 pandemic worsened this scenario, prompting the emergence of extremely resistant microorganisms. METHODS: Between March 2020 and September 2021, the NRL confirmed 82 clinical Enterobacterales isolates harboring a combination of blaKPC and MBL genes. Molecular typing was analyzed by PFGE and MLST. Modified double-disk synergy (MDDS) tests were used for phenotypic studies. RESULTS: Isolates were submitted from 28 hospitals located in seven provinces and Buenos Aires City, including 77 K. pneumoniae, 2 K. oxytoca, 2 C. freundii, and 1 E. coli. Almost half of K. pneumoniae isolates (n = 38; 49.4%), detected in 15 hospitals, belong to the CC307 clone. CC11 was the second clone, including 29 (37.7%) isolates (22, ST11 and 7, ST258) from five cities and 12 hospitals. Three isolates belonging to CC45 were also detected. The carbapenemase combinations observed were as follows: 55% blaKPC-2 plus blaNDM-5; 32.5% blaKPC-2 plus blaNDM-1; 5% blaKPC-3 plus blaNDM-1; 5% blaKPC-2 plus blaIMP-8; and 2.5% strain with blaKPC-2 plus blaNDM-5 plus blaOXA-163. Aztreonam/avibactam and aztreonam/relebactam were the most active combinations (100% and 91% susceptible, respectively), followed by fosfomycin (89%) and tigecycline (84%). CONCLUSIONS: The MDDS tests using ceftazidime-avibactam/EDTA and aztreonam/boronic acid disks improved phenotypic classification as dual producers. The successful high-risk clones of K. pneumoniae, such as hyper-epidemic CC307 and CC11 clones, drove the dissemination of double carbapenemase-producing isolates during the COVID-19 pandemic.

Prevalence of communicable and non-communicable diseases among adult immigrants from Venezuela at a university hospital in Colombia from 2017 to 2018.

Since 2015 immigration has increased significantly into Colombia. As immigrants who are not insured to the national health system present to public hospitals for medical care. However, there is little knowledge about the prevalence of communicable and non-communicable diseases amongst them. Ours was a cross-sectional study at a university hospital reviewing 154 medical records of Venezuelan immigrants treated by the Internal Medicine Specialty between 2017 & 2018. Non-communicable diseases representing 66.3% are the main cause of hospitalization, possibly owing to poor primary care.

Carbapenemase-Producing Extraintestinal Pathogenic Escherichia coli From Argentina: Clonal Diversity and Predominance of Hyperepidemic Clones CC10 and CC131.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes infections outside the intestine. Particular ExPEC clones, such as clonal complex (CC)/sequence type (ST)131, have been known to sequentially accumulate antimicrobial resistance that starts with chromosomal mutations against fluoroquinolones, followed with the acquisition of bla CTX-M-15 and, more recently, carbapenemases. Here we aimed to investigate the distribution of global epidemic clones of carbapenemase-producing ExPEC from Argentina in representative clinical isolates recovered between July 2008 and March 2017. Carbapenemase-producing ExPEC (n = 160) were referred to the Argentinean reference laboratory. Of these, 71 were selected for genome sequencing. Phenotypic and microbiological studies confirmed the presence of carbapenemases confirmed as KPC-2 (n = 52), NDM-1 (n = 16), IMP-8 (n = 2), and VIM-1 (n = 1) producers. The isolates had been recovered mainly from urine, blood, and abdominal fluids among others, and some were from screening samples. After analyzing the virulence gene content, 76% of the isolates were considered ExPEC, although non-ExPEC isolates were also obtained from extraintestinal sites. Pan-genome phylogeny and clonal analysis showed great clonal diversity, although the first phylogroup in abundance was phylogroup A, harboring CC10 isolates, followed by phylogroup B2 with CC/ST131, mostly H30Rx, the subclone co-producing CTX-M-15. Phylogroups D, B1, C, F, and E were also detected with fewer strains. CC10 and CC/ST131 were found throughout the country. In addition, CC10 nucleated most metalloenzymes, such as NDM-1. Other relevant international clones were identified, such as CC/ST38, CC155, CC14/ST1193, and CC23. Two isolates co-produced KPC-2 and OXA-163 or OXA-439, a point mutation variant of OXA-163, and three isolates co-produced MCR-1 among other resistance genes. To conclude, in this work, we described the molecular epidemiology of carbapenemase-producing ExPEC in Argentina. Further studies are necessary to determine the plasmid families disseminating carbapenemases in ExPEC in this region.

Malaria among migrants in a university hospital in Colombia during 2018: A case series.

Imported malaria has increased in Colombia since 2015 and has been attributed to migrants coming from Venezuela. We present a series of malaria cases, nested in a retrospective cross-sectional study between 2017 and 2018, aimed at calculating the prevalence of medical diseases among immigrants in a University Hospital in Colombia. Among 154 immigrants admitted for medical causes between 2017 and 2018, 8 were diagnosed with malaria, all due to Plasmodium vivax. Of these, seven had uncomplicated malaria, five had a previous history of malaria, one was critically ill, but none died. We highlight that, similar to other case series of imported malaria, Latin American migrants were young, with similar clinical profiles, having a low proportion of severe cases, and P. vivax was the most frequent cause.

Modulation of neutrophil extracellular traps release by Klebsiella pneumoniae.

One of the main bactericidal mechanisms of polymorphonuclear neutrophils (PMN) is the release of neutrophil extracellular traps (NETs), which capture and destroy pathogens. Klebsiella pneumoniae (Kpn) producer of carbapenemase (KPC) and belonging to the sequence type 258 (ST258), is a hyper epidemic clone that causes a large number of infections worldwide associated with high persistence and mortality. It is necessary to investigate the interaction of Kpn KPC with the immune system to improve prevention and treatment of infections mediated by this bacterium. Based on the hypothesis that Kpn is able to subvert PMN-mediated death, the aim was to assess whether Kpn KPC ST258 could modulate the bactericidal response of PMN, focusing on NETs formation, compared to another opportunistic pathogen, as Escherichia coli (Eco). The results showed that the release of NETs was absent when PMN were challenged with Kpn KPC, while Eco was a strong inducer of NETosis. Moreover, Kpn KPC was able to inhibit NETosis induced by Eco. The inhibition of Kpn KPC-mediated NETs formation still occurred in spite of exogenous addition of hydrogen peroxide (H2 O2 ), did not involve bacterial-released soluble factors or cell wall components, and was dependent on bacterial viability. Moreover, when degranulation was investigated, we found that Kpn KPC affected only the mobilization of primary granules, which harbor the proteins with more potent bactericidal properties and those related to NETosis. In conclusion, Kpn KPC ST258 effectively managed to evade the PMN response by inhibiting the release of NETs, and primary granule mobilization.

Comparative Kinetic Analysis of OXA-438 with Related OXA-48-Type Carbapenem-Hydrolyzing Class D ß-Lactamases.

Novel variants of OXA-48-type enzymes with the ability to hydrolyze oxyimino-cephalosporins and carbapenems are increasingly reported. Since its first report in 2011, OXA-163 is now extensively spread throughout Argentina, and several variants like OXA-247 have emerged. Here, we characterized a new blaOXA-48-like variant, OXA-438, and we performed a comparative kinetic analysis with the local variants OXA-247 and OXA-163 and the internationally disseminated OXA-48. blaOXA-163, blaOXA-247, and blaOXA-438 were located in a 70 kb IncN2 conjugative plasmid. OXA-438 presented mutations in the vicinity of conserved KTG (214-216), with a 2-aa deletion (R220-I221) and a D224E shift (as in OXA-163) compared to OXA-48. Despite Kpn163 (OXA-163), Kpn247 (OXA-247) and Eco438 (OXA-438) were resistant to meropenem and ertapenem, and the transconjugants (TC) remained susceptible (however, the carbapenems minimum inhibitory concentrations were ≥3 times 2-fold dilutions higher than the acceptor strain). TC163 and Eco48 were resistant to oxyimino-cephalosporins, unlike TC247 and TC438. kcat/Km values for cefotaxime in OXA-163 were slightly higher than the rest of the variants that were accompanied by a lower Km for carbapenems. For OXA-163, OXA-247, and OXA-438, the addition of NaHCO3 improved kcat values for both cefotaxime and ceftazidime; carbapenems kcat/Km values were higher than for oxyimino-cephalosporins. Mutations occurring near the conserved KTG in OXA-247 and OXA-438 are probably responsible for the improved carbapenems hydrolysis and decreased inactivation of oxyimino-cephalosporins compared to OXA-163. Dichroism results suggest that deletions at the ß5-ß6 loop seem to impact the structural stability of OXA-48 variants. Finally, additional mechanisms are probably involved in the resistance pattern observed in the clinical isolates.

Correction: Estimating the efficacy of community-wide use of systemic insecticides in dogs to control zoonotic visceral leishmaniasis: A modelling study in a Brazilian scenario.

[This corrects the article DOI: 10.1371/journal.pntd.0006797.].

Klebsiella pneumoniae ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

Estimating the efficacy of community-wide use of systemic insecticides in dogs to control zoonotic visceral leishmaniasis: A modelling study in a Brazilian scenario.

Systemic insecticides in dogs have been suggested as a public health intervention to prevent human cases of Zoonotic Visceral Leishmaniasis (ZVL). But, currently there are no systemic insecticides for dogs registered against zoo-anthropophilic pool blood feeding phlebotomine flies. We predict the impact of community-wide use of systemic insecticide in dog populations as a public health measure to control transmission of Leishmania infantum to humans using a mathematical model. We developed a Susceptible-Exposed-Infected (SEI) compartmental model to describe L. infantum transmission dynamics in dogs, with a vectorial capacity term to represent transmission between L. infantum-hosting dogs via phlebotomine flies. For Infected (I) dogs two levels of infectiousness were modelled, high infectiousness and low infectiousness. Human incidence was estimated through its relationship to infection in the dog population. We evaluated outcomes from a wide range of scenarios comprising different combinations of initial insecticide efficacy, duration of insecticide efficacy over time, and proportion of the dog population treated (60%, 70% & 80%). The same reduction in human infection incidence can be achieved via different combinations of insecticide efficacy, duration and dog coverage. For example, a systemic insecticide with an initial efficacy of 80% and 6 months above 65% efficacy would require treating at least 70% of the dogs to reduce the human infection incidence by 50%. Sensitivity analysis showed that the model outcome was most sensitive to baseline values of phlebotomine fly daily survival rate and insecticide coverage. Community-wide use of systemic insecticides applied to the "L. infantum canine reservoir" can significantly reduce human incidence of L. infantum infection. The results of this mathematical model can help defining the insecticide target product profile and how the insecticide should be applied to maximise effectiveness.

Genetic Diversity of KPC-Producing Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Citrobacter freundii Isolates from Argentina.

The predominance of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae was caused by the spread of ST258 clone. In Latin America, KPC was reported in 2006, with the isolation of genetically unrelated K. pneumoniae in Colombia. Since then, the expansion of blaKPC in either K. pneumoniae ST258 or other Enterobacteriaceae (ETB) species was increasingly reported. In this study, we characterized 89 KPC-producing Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Citrobacter freundii that were received between 2010 and 2014. The results revealed that all isolates harbored blaKPC-2. Moreover, the dissemination of KPC by non-K. pneumoniae was mainly caused by the dispersion of ETB mostly genetically unrelated. E. coli is a community pathogen that may serve as the vehicle for the spread of KPC into community settings. Recently, KPC was detected in E. coli ST131, an international epidemic and multidrug-resistant clone. We found that 5/29 KPC-producing E. coli belonged to ST131 and four were blaCTXM-15 producers. The detection of blaKPC in ST131 should be closely monitored to prevent further dissemination. The blaKPC is generally located within Tn4401 transposon capable of mobilization through transposition found in plasmids in ST258. Less is known about the diversity of blaKPC genetic elements that disseminate horizontally among other species of ETB. We found that 16/29 E. coli and 2/18 S. marcescens harbored blaKPC-2 in Tn4401a. In 71 isolates, blaKPC-2 was located amidst diverse Tn3-derived genetic elements bearing non-Tn4401 structure. Further studies on the plasmids that encode blaKPC-2 in these clinical isolates may provide additional insight into its transmission mechanisms.

Systemic insecticide treatment of the canine reservoir of Trypanosoma cruzi induces high levels of lethality in Triatoma infestans, a principal vector of Chagas disease.

BACKGROUND: Despite large-scale reductions in Chagas disease prevalence across Central and South America, Trypanosoma cruzi infection remains a considerable public health problem in the Gran Chaco region where vector-borne transmission persists. In these communities, peridomestic animals are major blood-meal sources for triatomines, and household presence of infected dogs increases T. cruzi transmission risk for humans. To address the pressing need for field-friendly, complementary methods to reduce triatomine infestation and interrupt T. cruzi transmission, this study evaluated the systemic activity of three commercial, oral, single dose insecticides Fluralaner (Bravecto®), Afoxolaner (NexGard®) and Spinosad (Comfortis®) in canine feed-through assays against Triatoma infestans, the principal domestic vector species in the Southern Cone of South America. METHODS: Twelve healthy, outbred dogs were recruited from the Zoonosis Surveillance and Control Program in Santa Cruz, Bolivia, and randomized to three treatment groups, each containing one control and three treated dogs. Following oral drug administration, colony-reared second and third stage T. infestans instars were offered to feed on dogs for 30 min at 2, 7, 21, 34 and 51 days post-treatment. RESULTS: Eighty-five per cent (768/907) of T. infestans successfully blood-fed during bioassays, with significantly higher proportions of bugs becoming fully-engorged when exposed to Bravecto® treated dogs (P < 0.001) for reasons unknown. Exposure to Bravecto® or NexGard® induced 100% triatomine mortality in fully- or semi-engorged bugs within 5 days of feeding for the entire follow-up period. The lethality effect for Comfortis® was much lower (50-70%) and declined almost entirely after 51 days. Instead Comfortis® treatment resulted in substantial morbidity; of these, 30% fully recovered whereas 53% remained morbid after 120 h, the latter subsequently unable to feed 30 days later. CONCLUSIONS: A single oral dose of Fluralaner or Afoxolaner was safe and well tolerated, producing complete triatomine mortality on treated dogs over 7.3 weeks. While both drugs were highly efficacious, more bugs exposed to Fluralaner took complete blood-meals, and experienced rapid knock-down. Coupled with its longer residual activity, Fluralaner represents an ideal insecticide for development into a complementary, operationally-feasible, community-level method of reducing triatomine infestation and potentially controlling T. cruzi transmission, in the Gran Chaco region.

Systemic insecticide treatment of the canine reservoir of Trypanosoma cruzi induces high levels of lethality in Triatoma infestans, a principal vector of Chagas disease

Background Despite large-scale reductions in Chagas disease prevalence across Central and South America, Trypanosoma cruzi infection remains a considerable public health problem in the Gran Chaco region where vector-borne transmission persists. In these communities, peridomestic animals are major blood-meal sources for triatomines, and household presence of infected dogs increases T. cruzi transmission risk for humans. To address the pressing need for field-friendly, complementary methods to reduce triatomine infestation and interrupt T. cruzi transmission, this study evaluated the systemic activity of three commercial, oral, single dose insecticides Fluralaner (Bravecto®), Afoxolaner (NexGard®) and Spinosad (Comfortis®) in canine feed-through assays against Triatoma infestans, the principal domestic vector species in the Southern Cone of South America. Methods Twelve healthy, outbred dogs were recruited from the Zoonosis Surveillance and Control Program in Santa Cruz, Bolivia, and randomized to three treatment groups, each containing one control and three treated dogs. Following oral drug administration, colony-reared second and third stage T. infestans instars were offered to feed on dogs for 30 min at 2, 7, 21, 34 and 51 days post-treatment. Results Eighty-five per cent (768/907) of T. infestans successfully blood-fed during bioassays, with significantly higher proportions of bugs becoming fully-engorged when exposed to Bravecto® treated dogs (P < 0.001) for reasons unknown. Exposure to Bravecto® or NexGard® induced 100% triatomine mortality in fully- or semi-engorged bugs within 5 days of feeding for the entire follow-up period. The lethality effect for Comfortis® was much lower (50­70%) and declined almost entirely after 51 days. Instead Comfortis® treatment resulted in substantial morbidity; of these, 30% fully recovered whereas 53% remained morbid after 120 h, the latter subsequently unable to feed 30 days later. Conclusions A single oral dose of Fluralaner or Afoxolaner was safe and well tolerated, producing complete triatomine mortality on treated dogs over 7.3 weeks. While both drugs were highly efficacious, more bugs exposed to Fluralaner took complete blood-meals, and experienced rapid knock-down. Coupled with its longer residual activity, Fluralaner represents an ideal insecticide for development into a complementary, operationally-feasible, community-level method of reducing triatomine infestation and potentially controlling T. cruzi transmission, in the Gran Chaco region.

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle.

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR=16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR=0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR=0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.

[Dissemination of blaIMP-8 among Enterobacteriaceae isolates from a Buenos Aires Hospital]. / Diseminación de blaIMP-8 en enterobacterias aisladas en un hospital de Buenos Aires.

From August 2008 to December 2011, six metallo-ß-lactamase-producing isolates, four Enterobacter cloacae, one Klebsiella oxytoca and one Citrobacter freundii, were detected at Hospital Interzonal General de Agudos "Evita" in Lanús. All six isolates showed multiresistant profiles and the presence of the blaIMP-8 gene. Five isolates also expressed PER-2 extended spectrum ß-lactamase. The blaIMP-8 gene was found as the first cassette in a class 1 integron. However, the 3´ conserved sequence could not be detected in three isolates. In all cases, blaIMP-8 was transferred by conjugation to azide-resistant Escherichia coli J53. PFGE analysis revealed that the four E. cloacae isolates were not genetically related. These are the first metallo-ß-lactamases detected in this institution and our results suggest a possible intra- and inter-species horizontal dissemination of blaIMP-8.

Diseminación de blaIMP-8 en enterobacterias aisladas en un hospital de Buenos Aires / [Dissemination of blaIMP-8 among Enterobacteriaceae isolates from a Buenos Aires Hospital].

From August 2008 to December 2011, six metallo-ß-lactamase-producing isolates, four Enterobacter cloacae, one Klebsiella oxytoca and one Citrobacter freundii, were detected at Hospital Interzonal General de Agudos "Evita" in Lanús. All six isolates showed multiresistant profiles and the presence of the blaIMP-8 gene. Five isolates also expressed PER-2 extended spectrum ß-lactamase. The blaIMP-8 gene was found as the first cassette in a class 1 integron. However, the 3´ conserved sequence could not be detected in three isolates. In all cases, blaIMP-8 was transferred by conjugation to azide-resistant Escherichia coli J53. PFGE analysis revealed that the four E. cloacae isolates were not genetically related. These are the first metallo-ß-lactamases detected in this institution and our results suggest a possible intra- and inter-species horizontal dissemination of blaIMP-8.

Model of polymicrobial peritonitis that induces the proinflammatory and immunosuppressive phases of sepsis.

Severe sepsis is associated with early release of inflammatory mediators that contribute to the morbidity and mortality observed during the first stages of this syndrome. Although sepsis is a deadly, acute disease, high mortality rates have been observed in patients displaying evidence of sepsis-induced immune deactivation. Although the contribution of experimental models to the knowledge of pathophysiological and therapeutic aspects of human sepsis is undeniable, most of the current studies using animal models have focused on the acute, proinflammatory phase. We developed a murine model that reproduces the early acute phases but also the long-term consequences of human sepsis. We induced polymicrobial acute peritonitis (AP) by establishing a surgical connection between the cecum and the peritoneum, allowing the exit of intestinal bacteria. Using this model, we observed an acute phase with high mortality, leukopenia, increased interleukin-6 levels, bacteremia, and neutrophil activation. A peak of leukocytosis on day 9 or 10 revealed the persistence of the infection within the lung and liver, with inflammatory hepatic damage being shown by histological examination. Long-term (20 days) derangements in both innate and adaptive immune responses were found, as demonstrated by impaired systemic tumor necrosis factor alpha production in response to an inflammatory stimulus; a decreased primary humoral immune response and T cell proliferation, associated with an increased number of myeloid suppressor cells (Gr-1(+) CD11b(+)) in the spleen; and a low clearance capacity. This model provides a good approach to attempt novel therapeutic interventions directed to augmenting host immunity during late sepsis.