A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples.

Introduction: Extended-spectrum ß-lactamase- (ESBL) and AmpC- ß-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development. Methods: Up to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three ß-lactamase groups. Results and discussion: The laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014-2027 (EU 2020/1729).

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance.

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.

Draft Genome Sequences of Lactococcus lactis Strains MS22314, MS22333, MS22336, and MS22337, Isolated from Fermented Camel Milk in Ethiopia.

The genome sequences of four Lactococcus lactis strains isolated from fermented camel milk were sequenced using paired-end Illumina MiSeq reads. The genome size of each strain was about 2.6 Mb, and three of the strains were annotated with tet(S) coding for tetracycline resistance.

Cross-Border Transmission of Salmonella Choleraesuis var. Kunzendorf in European Pigs and Wild Boar: Infection, Genetics, and Evolution.

Salmonella enterica subspecies enterica serotype Choleraesuis is a swine adapted serovar. S. Choleraesuis variant Kunzendorf is responsible for the majority of outbreaks among pigs. S. Choleraesuis is rare in Europe, although there have been serious outbreaks in pigs including two outbreaks in Denmark in 1999-2000 and 2012-2013. Here, we elucidate the epidemiology, possible transmission routes and sources, and clonality of European S. Choleraesuis isolates including the Danish outbreak isolates. A total of 102 S. Choleraesuis isolates from different European countries and the United States, covering available isolates from the last two decades were selected for whole genome sequencing. We applied a temporally structured sequence analysis within a Bayesian framework to reconstruct a temporal and spatial phylogenetic tree. MLST type, resistance genes, plasmid replicons, and accessory genes were identified using bioinformatics tools. Fifty-eight isolates including 11 out of 12 strains from wild boars were pan-susceptible. The remaining isolates carried multiple resistance genes. Eleven different plasmid replicons in eight plasmids were determined among the isolates. Accessory genes were associated to the identified resistance genes and plasmids. The European S. Choleraesuis was estimated to have emerged in ∼1837 (95% credible interval, 1733-1983) with the mutation rate of 1.02 SNPs/genome/year. The isolates were clustered according to countries and neighbor countries. There were transmission events between strains from the United States and European countries. Wild boar and pig isolates were genetically linked suggesting cross-border transmission and transmission due to a wildlife reservoir. The phylogenetic tree shows that multiple introductions were responsible for the outbreak of 2012-2013 in Denmark, and suggests that poorly disinfected vehicles crossing the border into Denmark were potentially the source of the outbreak. Low levels of single nucleotide polymorphisms (SNPs) differences (0-4 SNPs) can be observed between clonal strains isolated from different organs of the same animal. Proper disinfection of livestock vehicles and improved quality control of livestock feed could help to prevent future spread of S. Choleraesuis or other more serious infectious diseases such as African swine fever (ASF) in the European pig production system.

Direct whole-genome sequencing of Plasmodium falciparum specimens from dried erythrocyte spots.

BACKGROUND: Plasmodium falciparum malaria remains a major health burden and genomic research represents one of the necessary approaches for continued progress towards malaria control and elimination. Sample acquisition for this purpose is troublesome, with the majority of malaria-infected individuals living in rural areas, away from main infrastructure and the electrical grid. The aim of this study was to describe a low-tech procedure to sample P. falciparum specimens for direct whole genome sequencing (WGS), without use of electricity and cold-chain. METHODS: Venous blood samples were collected from malaria patients in Bandim, Guinea-Bissau and leukocyte-depleted using Plasmodipur filters, the enriched parasite sample was spotted on Whatman paper and dried. The samples were stored at ambient temperatures and subsequently used for DNA-extraction. Ratios of parasite:human content of the extracted DNA was assessed by qPCR, and five samples with varying parasitaemia, were sequenced. Sequencing data were used to analyse the sample content, as well as sample coverage and depth as compared to the 3d7 reference genome. RESULTS: qPCR revealed that 73% of the 199 samples were applicable for WGS, as defined by a minimum ratio of parasite:human DNA of 2:1. WGS revealed an even distribution of sequence data across the 3d7 reference genome, regardless of parasitaemia. The acquired read depths varied from 16 to 99×, and coverage varied from 87.5 to 98.9% of the 3d7 reference genome. SNP-analysis of six genes, for which amplicon sequencing has been performed previously, confirmed the reliability of the WGS-data. CONCLUSION: This study describes a simple filter paper based protocol for sampling P. falciparum from malaria patients for subsequent direct WGS, enabling acquisition of samples in remote settings with no access to electricity.

Extended-spectrum-beta-lactamases, AmpC beta-lactamases and plasmid mediated quinolone resistance in klebsiella spp. from companion animals in Italy.

We report the genetic characterization of 15 Klebsiella pneumoniae (KP) and 4 isolates of K. oxytoca (KO) from clinical cases in dogs and cats and showing extended-spectrum cephalosporin (ESC) resistance. Extended spectrum beta-lactamase (ESBL) and AmpC genes, plasmid-mediated quinolone resistance (PMQR) and co-resistances were investigated. Among KP isolates, ST101 clone was predominant (8/15, 53%), followed by ST15 (4/15, 27%). ST11 and ST340, belonging to Clonal Complex (CC)11, were detected in 2012 (3/15, 20%). MLST on KP isolates corresponded well with PFGE results, with 11 different PFGE patterns observed, including two clusters of two (ST340) and four (ST101) indistinguishable isolates, respectively. All isolates harbored at least one ESBL or AmpC gene, all carried on transferable plasmids (IncR, IncFII, IncI1, IncN), and 16/19 were positive for PMQR genes (qnr family or aac(6')-Ib-cr). The most frequent ESBL was CTX-M-15 (11/19, 58%), detected in all KP ST101, in one KP ST15 and in both KP ST340. blaCTX-M-15 was carried on IncR plasmids in all but one KP isolate. All KP ST15 isolates harbored different ESC resistance genes and different plasmids, and presented the non-transferable blaSHV-28 gene, in association with blaCTX-M-15, blaCTX-M-1 (on IncR, or on IncN), blaSHV-2a (on IncR) or blaCMY-2 genes (on IncI1). KO isolates were positive for blaCTX-M-9 gene (on IncHI2), or for the blaSHV-12 and blaDHA-1 genes (on IncL/M). They were all positive for qnr genes, and one also for the aac(6')-Ib-cr gene. All Klebsiella isolates showed multiresistance towards aminoglycosides, sulfonamides, tetracyclines, trimethoprim and amphenicols, mediated by strA/B, aadA2, aadB, ant (2")-Ia, aac(6')-Ib, sul, tet, dfr and cat genes in various combinations. The emergence in pets of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bi-directional transmission between pets and humans, especially at household level.

Rapid whole-genome sequencing for detection and characterization of microorganisms directly from clinical samples.

Whole-genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples, this could further reduce diagnostic times and thereby improve control and treatment. A major bottleneck is the availability of fast and reliable bioinformatic tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatic tools for the analysis of sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria, and direct sequencing on pellets from the urine samples. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples but in pure cultures from only 17 samples. WGS improved the identification of the cultivated bacteria, and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibilities. Complete agreement was observed between species identification, multilocus sequence typing, and phylogenetic relationships for Escherichia coli and Enterococcus faecalis isolates when the results of WGS of cultured isolates and urine samples were directly compared. Sequencing directly from the urine enabled bacterial identification in polymicrobial samples. Additional putative pathogenic strains were observed in some culture-negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic times. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem, a publicly available bioinformatic tool was developed in this study.

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

INTRODUCTION: Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. METHODOLOGY: Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. RESULTS: All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. CONCLUSION: We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

Occurrence of Listeria spp. in retail meat and dairy products in the area of Addis Ababa, Ethiopia.

BACKGROUND: Listeriosis, a bacterial disease in humans and animals, is mostly caused by ingestion of Listeria monocytogenes via contaminated food and/or water, or by a zoonotic infection. Globally, listeriosis has in general a low incidence but a high case fatality rate. OBJECTIVE: The objective of this study was to investigate the occurrence, antimicrobial profiles, and genetic relatedness of L. monocytogenes from raw meat and dairy products (raw milk, cottage cheese, cream cake), collected from the capital and five neighboring towns in Ethiopia. METHODS: Two hundred forty food samples were purchased from July to December 2006 from food vendors, shops, and supermarkets, using a cross-sectional study design. L. monocytogenes were isolated and subjected to molecular serotyping. The genetic relatedness and antimicrobial susceptibility patterns were investigated using pulsed-field gel electrophoresis (PFGE) and minimum inhibitory concentration determinations. RESULTS: Of 240 food samples tested, 66 (27.5%) were positive for Listeria species. Of 59 viable isolates, 10 (4.1%) were L. monocytogenes. Nine were serotype 4b and one was 2b. Minimum inhibitory concentration determination and PFGE of the 10 L. monocytogenes isolates showed low occurrence of antimicrobial resistance among eight different PFGE types. DISCUSSION AND CONCLUSIONS: The findings in this study correspond to similar research undertaken in Ethiopia by detecting L. monocytogenes with similar prevalence rates. Public education is crucial as regards the nature of this organism and relevant prevention measures. Moreover, further research in clinical samples should be carried out to estimate the prevalence and carrier rate in humans, and future investigations on foodborne outbreaks must include L. monocytogenes.

Molecular clonality and antimicrobial resistance in Salmonella enterica serovars Enteritidis and Infantis from broilers in three Northern regions of Iran.

BACKGROUND: Multidrug-resistant Salmonella strains are frequently encountered problems worldwide with considerable increased occurrences in recent years. The aim of this study was to investigate the occurrence and frequency of antimicrobial resistance and associated resistance genes in Salmonella isolates from broiler farms in different regions of Iran covering a time period of four years. RESULTS: From 2007 to 2011, 36 Salmonella strains were isolated from broiler farms located in three northern provinces of Iran. The isolates were serotyped, antimicrobial susceptibility tested, and characterized for antimicrobial resistance genes associated to the phenotype. Pulsed-field gel electrophoresis (PFGE) was applied for comparison of genetic relatedness.Two serovars were detected among the isolates; Salmonella enterica serovar Infantis (75%) and S. Enteritidis (25%). Thirty-four (94%) of the isolates exhibited resistance to nalidixic acid and ciprofloxacin caused by a single mutation in the quinolone resistance-determining region (QRDR) of gyrA. For all strains this mutation occurred in the codon of Asp87 leading to a Asp87-Tyr, Asp87-Gly or Asp87-Asn substitutions. All S. Infantis (n = 27) were resistant to tetracycline, spectinomycin, streptomycin, and sulfamethoxazole and harbored the associated resistance genes; tetA, dfrA14, aadA1, and sulI together with class 1 integrons. The isolates revealed highly similar PFGE patterns indicating clonal relatedness across different geographical locations. CONCLUSION: The data provided fundamental information applicable when launching future control programs for broilers in Iran with the aim to conserve the effectiveness of important antimicrobials for treatment in humans.

Characterization of Salmonella enterica serovar Enteritidis isolates recovered from blood and stool specimens in Thailand.

BACKGROUND: Bacteremia due to Salmonella spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal Salmonella (NTS) serovars isolated from Thai patients between 2002-2007. In this study, 60.8% of Salmonella enterica serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA. RESULT: Eight antibiogrammes, seven MLVA types, 14 XbaI/BlnI PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant. CONCLUSIONS: The increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.

SCCmec Type IX Element in Methicillin Resistant Staphylococcusaureusspa Type t337 (CC9) Isolated from Pigs and Pork in Thailand.

Methicillin resistant Staphylococcusaureus (MRSA) have emerged among livestock in several countries. In this study, we describe the results of a screening performed in pigs and raw pork samples in Thailand. Ten pork samples and 15 nasal swabs from pigs were collected from 2 markets and 1 pig farm in the Samuth Songkhram province in Thailand. MRSA were isolated using selective isolation procedures and confirmed by mecA PCR. The MRSA were characterized by antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE), spa typing, SCCmec typing, and MLST. Resistance and virulence markers were screened using a microarray. Five of the pork samples and six pig nasal swabs were positive for MRSA. All 11 isolates belonged to spa type t337 but showed diversity in antimicrobial resistance patterns and PFGE profiles. Additionally, the isolates were sequence-typed; ST9, ST2136, ST2278 belonging to the clonal complex; CC9. All isolates harbored SCCmec IX and were resistant to 7 out of 14 tested antimicrobials; additional resistances to all antimicrobials tested were found in some of the pork and pig isolates and 1 pork isolate was resistant to 13 antimicrobials tested. Microarray analysis identified blaZ, aac-aphD, vga(A), tetM, and a tet efflux marker, in all strains and additionally ermB and aadD, cat and fex(A) in the pork isolates. None of the isolates were found PVL-positive, but enterotoxins were identified in all isolates. To our knowledge, only a few descriptions of MRSA in livestock and food products in Thailand have been observed but this is the first observation of MRSA CC9 associated with SCCmec IX in pork. This study indicates a likely widespread distribution of MRSA in pig and pork in Thailand and further investigation on the prevalence and importance of livestock associated MRSA in Thailand is needed.

Antimicrobial-resistant Shigella infections from Iran: an overlooked problem?

OBJECTIVES: In this study, we wanted to assess the level of antimicrobial resistance, the presence of genes encoding resistance to cephalosporins and plasmid-mediated quinolone resistance (PMQR), and genetic relatedness among Shigella isolates obtained from Iranian patients. METHODS: A total of 44 Shigella isolates were collected from Iranian patients admitted to Milad Hospital, Tehran, Iran, during 2008-10. Of these, 37 were serotyped and characterized by MIC determination. A subset of eight suspected extended-spectrum ß-lactamase (ESBL) producers (six Shigella sonnei phase II and two Shigella flexneri type 1b) were examined for the presence of genes encoding cephalosporin resistance. The presence of PMQR was assessed in one S. flexneri isolate exhibiting low-level resistance to ciprofloxacin and susceptibility to nalidixic acid. PFGE was performed on 25 S. sonnei phase II isolates. RESULTS: Of the isolates, 25 (68%) were S. sonnei phase II, with 5 (14%) S. flexneri, 5 (14%) Shigella dysenteriae type 2, and 2 (5%) Shigella boydii type 2. Resistance to at least threeclasses of antimicrobials was detected in all species. The presence of bla(CTX-M-15) and the AmpC ß-lactamase producer bla(CMY-2) was confirmed in five and one S. sonnei phase II isolates, respectively. One of the two S. flexneri type 1b that contained bla(CTX-M-15) also harboured a qnrS1 gene. PFGE identified sevenPFGE profiles; the main cluster included 15 of the strains, suggesting low genetic diversity between isolates or the presence of an endemic clone in Iran. CONCLUSIONS: This is the first known description of ESBL-producing and AmpC ß-lactamase-producing Shigella and of PMQR Shigella in Iran. The emergence of CTX-15, CMY-2 and qnrS1 genes may compromise the treatment of shigellosis. Strategies to minimize the spread of ESBL-producing and AmpC-ß-lactamase-producing Shigella should be implemented.

Characterization of isolates of Salmonella enterica serovar Stanley, a serovar endemic to Asia and associated with travel.

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding bla(CMY-2) gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad.