Ma Argudín

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

ABSTRACT Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

Extended-spectrum β-lactamase- and AmpC β-lactamase-producing d-tartrate-positive Salmonella enterica serovar Paratyphi B from broilers and human patients in Belgium, 2008–10

OBJECTIVES To characterize the genetic determinants responsible for extended-spectrum cephalosporin (ESC) resistance of d-tartrate-positive Salmonella enterica subsp. enterica serovar Paratyphi B (serovar Paratyphi B dT+) strains that have emerged in poultry and humans in Belgium during 2008-10. METHODS The ESC resistance genes among non-redundant serovar Paratyphi B dT+ strains were determined using PCR and sequencing. ESC phenotypes were horizontally transferred by conjugation. Extended-spectrum β-lactamase (ESBL)- or AmpC-carrying plasmids were typed by PCR-based replicon typing, plasmid multilocus sequence typing and restriction fragment length polymorphism. The genetic relationship of ESC-resistant strains was assessed by XbaI PFGE and multilocus sequence typing. RESULTS Since 2008, the proportion of serovar Paratyphi B dT+ strains from broiler origin has increased significantly to reach 36.5% in 2010. Among 95 non-duplicate serovar Paratyphi B dT+ strains, 35% were resistant to ESCs. At the same time, a few ESC-resistant serovar Paratyphi B dT+ strains from humans were also detected in Belgium. The most prevalent ESBL gene, blaCTX-M-1, and the AmpC cephalosporinase gene blaCMY-2 were identified on various conjugative IncI1 plasmids of different sequence types and with different additional non-β-lactam phenotypes. Interestingly, the blaCTX-M-2 gene was located on large multireplicon IncHI2/P plasmids. In addition, highly ESC-resistant strains contained both the ESBL CTX-M-2 and the AmpC CMY-2 encoded by the IncHI2/P and IncI1 plasmids, respectively. All ESC-resistant serovar Paratyphi B dT+ strains belonged to sequence type 28 and showed the common PFGE pattern X8, as well as the chromosomal class 2 integron cassette array dfrA1-sat2-aadA1 previously described in the European poultry-associated serovar Paratyphi B dT+ clonal population. CONCLUSIONS This study showed that the clonal population of multidrug-resistant serovar Paratyphi B dT+, persisting in broilers in Belgium for the last decade, recently acquired various plasmid-borne ESC resistance determinants, constituting a major concern for public health. Further surveillance programmes and research are an absolute necessity to understand their epidemiology and to propose interventions to limit the spread of ESC- and multidrug-resistant Salmonella spp.

Characterization of methicillin-resistant Staphylococcus sciuri isolates from industrially raised pigs, cattle and broiler chickens

OBJECTIVES This study aimed at assessing the epidemiology and genetic diversity of methicillin-resistant Staphylococcus sciuri (MRSS) from different farm animal species. METHODS Nasal swabs were collected from 200 pigs, 100 dairy cows, 100 beef cows, 150 veal calves and 200 broilers. Colonies were isolated on selective media containing cefoxitin and the mecA gene was detected by PCR. Antimicrobial resistance was determined by broth microdilution. The genetic diversity was assessed by PFGE and resistance and virulence genes were detected by microarray analysis. RESULTS The total MRSS prevalence at the animal level was estimated at 9.5%, varying from ∼10% in veal (13.3%), broilers (12.5%) and dairy cows (10.0%) to 6.5% in pigs and 3.0% in beef cows. mecA was detected in all isolates. SCCmec elements of type III and non-typeable ones were seen most frequently. More than 90% of isolates were non-wild-type (NWT) for gentamicin, penicillin, tiamulin, clindamycin and quinupristin/dalfopristin. The frequency of NWT isolates for fusidic acid and trimethoprim ranged between 78% and 87%. PFGE analysis allowed distinction between two major clusters. Most isolates tested by microarray carried erm and tet genes. Virulence genes were also detected, including an isa gene encoding an immune-evasion factor and the hsdS2 gene encoding a site-specific deoxyribonuclease. CONCLUSIONS This study shows that multiresistant MRSS is carried by different farm animal species. Although some animals shared the same strain, PFGE showed different patterns, indicating high diversity among the MRSS isolates recovered. The absence of clusters associated with a certain animal species suggests low host specificity.

Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital

ABSTRACT Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus (n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR (n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR34/L98H was the most prevalent mutation (46.7%), followed by TR46/Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

Bacteria from Animals as a Pool of Antimicrobial Resistance Genes

Antimicrobial agents are used in both veterinary and human medicine. The intensive use of antimicrobials in animals may promote the fixation of antimicrobial resistance genes in bacteria, which may be zoonotic or capable to transfer these genes to human-adapted pathogens or to human gut microbiota via direct contact, food or the environment. This review summarizes the current knowledge of the use of antimicrobial agents in animal health and explores the role of bacteria from animals as a pool of antimicrobial resistance genes for human bacteria. This review focused in relevant examples within the ESC(K)APE (Enterococcus faecium, Staphylococcus aureus, Clostridium difficile (Klebsiella pneumoniae), Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae) group of bacterial pathogens that are the leading cause of nosocomial infections throughout the world.

In vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during a national survey conducted in Belgian hospitals

Objectives The aim of this study was to estimate the in vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during national surveillance in Belgian acute-care hospitals. Ceftaroline-resistant isolates were further investigated for their resistance mechanisms. Methods From October 2013 to March 2014, 155 laboratories of Belgian acute-care hospitals were invited to send to the National Reference Centre—Staphylococcus aureus (Belgium) up to five non-duplicate S. aureus including three MRSA and two MSSA from hospitalized patients. Isolates were analysed by spa typing, SCCmec typing (for MRSA) and PCR for detection of 16S-mecA-nuc and 16S-mecC. MICs of oxacillin, cefoxitin and ceftaroline were determined by the broth microdilution method. The nucleotide sequences of mecA, native pbp and gdpP genes of isolates with reduced susceptibility to ceftaroline were analysed for the presence of mutations responsible for amino acid substitutions. Results Ninety-nine percent of isolates, including MRSA (n = 284) and MSSA (n = 131), were susceptible to ceftaroline. Only four MRSA isolates showed resistance to ceftaroline (MIC = 2 mg/L). These four isolates belonged to lineages CC5 (n = 1), CC22 (n = 2) and CC8 (n = 1). Two isolates (CC22 and CC8) carried mutations in mecA, as well as in other pbp genes. The remaining isolates carried mutations in native pbp genes or in gdpP. Conclusions This is the first Belgian in vitro survey on ceftaroline activity against S. aureus. This antibiotic showed excellent activity against MRSA and MSSA, and only a few MRSA isolates with resistance were found. Reduced susceptibility to ceftaroline seems a complex phenomenon due to the accumulation of mutations in genes involved in &bgr;-lactam tolerance.

Clinical case of cfr-positive MRSA CC398 in Belgium.

Sir, Linezolid, the first member of the oxazolidinone class of antibiotics, is one of the major antimicrobial agents used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. This drug inhibits bacterial protein synthesis by binding to domain Vof the 23S ribosomal RNA (rRNA) of the 50S subunit. Several mechanisms conferring linezolid resistance have been described in staphylococci, including point mutations in genes encoding 23S rRNA and mutations in ribosomal proteins L3, L4 and L22 [1, 2]. Even more problematic is the identification of cfr, a horizontally transferable resistance gene that encodes a ribosomal methyltransferase conferring cross-resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A (PhLOPSA phenotype) [1, 2]. Recently, a new gene (optrA) coding for an ABC transporter which confers resistance to oxazolidinones and phenicol resistance has been described. Yet, this new gene has only been found in enterococci and S. sciuri [3, 4]. Despite its clinical use for more than 15 years, linezolid resistance remains scarce and is mostly due to the presence of chromosomal mutations. A recent study regarding the continuous long-term and stable in vitro activity of linezolid against different bacteria (including S. aureus, coagulasenegative staphylococci, Enterococcus spp. and Streptococcus spp.) [5] showed that none of the 7541 collected organisms harboured the cfr gene. However, wewere recently confronted with the management of one case of a human infection caused by a cfr-positive MRSA belonging to the livestock-associated (LA-) clone ST398, which is, to the best of our knowledge, the first clinical case reported in Belgium. A 74-year-old man was admitted to hospital for a postoperative skin and soft tissue infection complicating a left carotid endarterectomy performed 2 weeks earlier. Cultures of the purulent discharge grew mixed abundant organisms consisting of Escherichia coli and an MRSA. Amoxicillin– clavulanic acid was the only antimicrobial agent administered in the last several years. After surgical drainage, antimicrobial therapy was started with intravenous vancomycin to achieve serum concentrations of 20 to 30 mg/L plus piperacillin–tazobactam (4/0.5 g q.i.d.). The therapy was switched after 6 days to an oral combination of cefuroxime axetil 500 mg t.i.d. and trimethoprim–sulphamethoxazole 160/800 mg b.i.d. for a total period of 14 days. This medico-surgical management lead to total resolution and cure of infection. As the MRSA isolate collected from the patient’s sample was tested to be resistant to linezolid by the VITEK 2 system, it was sent for confirmation to the National Reference Centre (NRC) for staphylococci. At the NRC, the identification was further confirmed genotypically by polymerase chain reaction (PCR) for the detection of 16S, mecA and nuc genes, as previously described [6]. Isolates were further tested by multilocus sequence typing (MLST) (http://saureus.mlst. net/). Antimicrobial susceptibility was determined by the disc diffusion method for the following antibiotics: cefoxitin, gentamicin, kanamycin, tobramycin, fusidic acid, * H. Paridaens henry.paridaens@gmail.com

Introduction to Antimicrobial-Resistant Foodborne Pathogens

The discovery of antimicrobial agents in the mid-twentieth century revolutionized the management and treatment of bacterial infections. Infections that would normally have been fatal became curable. Ever since then, antimicrobial agents have saved the lives of millions of people. These gains are now seriously jeopardized by the rapid emergence and spread of microbes that are resistant to antimicrobials. Moreover, the emergence of antimicrobial resistance is accompanied with a decline in the discovery of new antimicrobial agents. We are facing the possibility of a future without effective antibiotics for some infections and a scenario where infections that hitherto were considered harmless are now a serious health problem and a major cause of morbidity and mortality, together with major financial and social repercussions. In this chapter, the term antimicrobial resistance will be defined, and the ways it spreads among foodborne pathogens will be described.

Nosocomial Intravascular Catheter Infections with Extended-spectrum Beta-lactamase-producing Escherichia coli in Calves after Strain Introduction from a Commercial Herd

&NA; An outbreak of intravascular catheter‐related infections by extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in calves in an animal teaching hospital is reported. Pulsed‐field gel electrophoresis was used for strain typing to determine the origin and dissemination of these strains. All 19 strains harboured the blaCTX‐M‐14, and six strains also overexpressed their chromosomal AmpC gene. Evidence on the introduction of the strain from a beef herd, experiencing neonatal diarrhoea and increased mortality, to the clinic through admission of diarrhoeic calves was provided. Strains isolated from phlebitis cases from other herds up to 5 months later showed a high similarity with the initial strain, suggesting that the strain had become nosocomial. The catheter infections with ESBL/AmpC‐producing E. coli resulted in a prolonged hospitalization, increased anti‐microbial use and mortality. This report points towards the potential dangers of the emergence of ESBL/AmpC‐producing bacteria in susceptible food animals and warns farmers and veterinarians for the facility by which they are introduced into another environment.