Immune Evasion Cluster Genes Research Articles

Comparative genomics of Staphylococcus aureus strains from wild birds and pig farms elucidates levels of mobilomes, antibiotic pressure and host adaptation

This study characterized the resistome, mobilome and phylogenomic relatedness of Staphylococcus aureus strains previously obtained from healthy nestling storks (HNS), pigs (HP) and pig farmers (HPF) to analyse possible transmission pathways of S. aureus with implications for the spread of antimicrobial resistance. The genomic contents of 52 S. aureus strains obtained from the nasal cavity of HNS, HP and HPF in Spain were sequenced using the Illumina NextSeq platform to characterize their resistome, virulome and mobile genetic elements. The relatedness of strains was assessed by core-genome single nucleotide polymorphisms (SNPs). The frequencies of multidrug-resistance phenotype and transposons were significantly lower in strains from HNS than in those from HP and HPF (P < 0.005). However, the presence of human immune evasion cluster genes in S. aureus strains from HNS was significantly higher than in those from HP and HPF (P < 0.005). Interestingly, the frequencies of plasmids and phages were not significantly associated with the host (P > 0.05). The phylogenetic analysis identified a cluster of all the MSSA-CC398 strains carrying φSa3 and ermT on rep13 separately from the two MRSA-CC398 strains (carrying ermT on repUS18). Highly related MRSA-CC398 strains were detected in some pigs and related farmers (<10 SNPs). This study confirms high-level antibiotic selection in S. aureus in HP and HPF in comparison to HNS. Furthermore, our findings highlight the continuous transmission of MRSA-CC398 in the pig-to-human interface and MSSA-CC398 with human adaptation markers in HNS. Molecular surveillance of S. aureus using the One Health model is required to establish appropriate control strategies.

Exploring the virulence potential of immune evasion cluster genes in methicillin-resistant Staphylococcus aureus from cancer patients

Methicillin-resistant Staphylococcus aureus (MRSA) is accountable for a plethora of infections, ranging from minor cutaneous manifestations to grave metastatic conditions. The dissemination of MRSA among cancer patients poses a substantial public health hazard on a global scale. This study explores the association between MRSA and bacteriophage-encoded immune evasion cluster (IEC) genes. This investigation employed a total of 168 pathogenic MRSA collected from 38 cancer and 130 non-cancer patients. A cefoxitin disc diffusion method followed by PCR analysis was used to identify the mecA gene. In this study, we employed singleplex and multiplexed PCR techniques to detect specific IEC genes. No association (p = 0.98) was observed between the sex and age of patients and MRSA isolates. However, MRSA isolates demonstrated a notable association (p = 0.01) with pus samples in non-cancer patients and skin swabs in cancer patients. The resistance profiles of MRSA strains from cancer and non-cancer patients did not show significant differences (p > 0.05). Notably, the sea gene was found to be more prevalent in MRSA isolates from cancer patients, displaying a significant association (p = 0.03). Additionally, this study identified two novel and distinct combinations of IEC types, namely V1 (sea, chp, scn) and V2 (sea, scn). Cancer patients had higher multidrug resistance and toxin gene abundance than non-cancer patients. The identification of two novel IEC patterns underscores the urgent need to control MRSA dissemination in hospitals and monitor emerging clones.

Genomic analysis of Staphylococcus aureus sequential isolates from lungs of patients with cystic fibrosis

Staphylococcus aureus is the predominant pathogen in children with cystic fibrosis (CF) in France and, around 80% of them harbored S. aureus in their lungs. This study investigated virulence and antimicrobial resistance-associated genes and within-host evolution polymorphisms in 14 S. aureus persistent clones from 14 chronically infected CF children. For each of the 14 patients, we compared genomes of two isogenic sequential isolates separated by 2–9 years. All isolates were methicillin-sensitive and harbored the immune evasion gene cluster, whereas half of them harbored the enterotoxin gene cluster. Most clones were capsule type 8 (8/14) and accessory gene regulator (agr)-specificity group 1 (9/14). We identified convergent mutations in genes involved in carbohydrate metabolism, cell wall metabolism, genetic information processing and adhesion, which are likely to play important role in intracellular invasion and persistence. Further explorations relying notably on proteomics will contribute to improve our understanding of the mechanisms at play in the striking long-term persistence ability of S. aureus.

Clonal dissemination of successful emerging clone mecA-MRSA t304/ST6 among humans and hedgehogs in the Helsinki metropolitan area in Finland

Methicillin-resistant Staphylococcus aureus (MRSA) carrying mecC gene (mecC-MRSA) is frequently reported among European hedgehogs (Europeaus erineaus) due to co-evolutionary adaptation to dermatophyte infection in European hedgehogs. The occurrence of MRSA in European hedgehogs in Finland is unknown. Consequently, we investigated the occurrence of MRSA in wild hedgehogs from urban Helsinki metropolitan area in 2020–2021 and applied whole genome sequencing (WGS) to further characterize the studied isolates and compared them with human clinical MRSA isolates. Altogether 115 dead hedgehogs were screened for MRSA using selective cultivation methods. Presumptive MRSA isolates were tested for antimicrobial susceptibility and confirmed MRSA isolates were further characterized by spa-typing and WGS. Hedgehog derived MRSA isolates were compared with clinical human MRSA isolates using core genome multilocus sequence typing (cgMLST). In total MRSA was recovered from 11 out of 115 (10%) hedgehogs. Among these four different spa types (t304; n = 4, t8835; n = 4, t5133; n = 2 and t622; n = 1) and three different sequence types (STs) (ST6; n = 6, ST7663; n = 4 and ST2840; n = 1) were identified. From the studied MRSA isolates seven harboured the mecA gene (mecA-MRSA) and four were identified as mecC-MRSA. All mecA-MRSA isolates carried immune evasion cluster genes, and one isolate was positive for Panton-Valentine leukocidin. cgMLST comparison revealed close genetic relatedness among three hedgehog and two human mecA-MRSA isolates all belonging to t304/ST6. Our results suggest a clonal dissemination of a successful MRSA clone among humans and hedgehogs. Further studies are warranted to investigate the sources and dissemination of such clone in urban environments. We observed a relatively low occurrence of mecC-MRSA in Finnish hedgehogs.

Stable antibiotic resistance and rapid human adaptation in livestock-associated MRSA.

Mobile genetic elements (MGEs) are agents of horizontal gene transfer in bacteria, but can also be vertically inherited by daughter cells. Establishing the dynamics that led to contemporary patterns of MGEs in bacterial genomes is central to predicting the emergence and evolution of novel and resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex (CC) 398 is the dominant MRSA in European livestock and a growing cause of human infections. Previous studies have identified three categories of MGEs whose presence or absence distinguishes livestock-associated CC398 from a closely related and less antibiotic-resistant human-associated population. Here, we fully characterise the evolutionary dynamics of these MGEs using a collection of 1180 CC398 genomes, sampled from livestock and humans, over 27 years. We find that the emergence of livestock-associated CC398 coincided with the acquisition of a Tn916 transposon carrying a tetracycline resistance gene, which has been stably inherited for 57 years. This was followed by the acquisition of a type V SCCmec that carries methicillin, tetracycline, and heavy metal resistance genes, which has been maintained for 35 years, with occasional truncations and replacements with type IV SCCmec. In contrast, a class of prophages that carry a human immune evasion gene cluster and that are largely absent from livestock-associated CC398 have been repeatedly gained and lost in both human- and livestock-associated CC398. These contrasting dynamics mean that when livestock-associated MRSA is transmitted to humans, adaptation to the human host outpaces loss of antibiotic resistance. In addition, the stable inheritance of resistance-associated MGEs suggests that the impact of ongoing reductions in antibiotic and zinc oxide use in European farms on livestock-associated MRSA will be slow to be realised.

Antimicrobial Susceptibility Profiles and Molecular Characterisation of Staphylococcus aureus from Pigs and Workers at Farms and Abattoirs in Zambia.

Pigs have been shown to be a reservoir for recently emerging livestock-associated Staphylococcus aureus (LA-SA), including methicillin resistant strains in many countries worldwide. However, there is sparse information about LA-SA strains circulating in Zambia. This study investigated the prevalence, phenotypic and genotypic characteristics of S. aureus from pigs and workers at farms and abattoirs handling pigs in Lusaka Province of Zambia. A total of 492 nasal pig swabs, 53 hand and 53 nasal human swabs were collected from farms and abattoirs in selected districts. Standard microbiological methods were used to isolate and determine antimicrobial susceptibility patterns of S. aureus. Polymerase Chain Reaction was used to confirm the species identity and detect antimicrobial resistance and virulence genes of isolates, whereas genetic diversity was evaluated using spa typing. Overall prevalence of S. aureus was 33.1%, 37.8% for pigs and 11.8% for humans. The isolates were resistant to several antibiotics with resistance ranging from 18% to 98% but were all susceptible to vancomycin. Typical LA-SA spa types were detected. The presence of plasmid mediated resistance genes such as tetM (12.8%), other resistance determinants and immune evasion cluster genes among the isolates is of great public health concern. Thus, continuous surveillance of S. aureus using a “One health” approach is warranted to monitor S. aureus infections and spread of antimicrobial resistance.

Prophages encoding human immune evasion cluster genes are enriched in Staphylococcus aureus isolated from chronic rhinosinusitis patients with nasal polyps.

Prophages affect bacterial fitness on multiple levels. These include bacterial infectivity, toxin secretion, virulence regulation, surface modification, immune stimulation and evasion and microbiome competition. Lysogenic conversion arms bacteria with novel accessory functions thereby increasing bacterial fitness, host adaptation and persistence, and antibiotic resistance. These properties allow the bacteria to occupy a niche long term and can contribute to chronic infections and inflammation such as chronic rhinosinusitis (CRS). In this study, we aimed to identify and characterize prophages present in Staphylococcus aureus from patients suffering from CRS in relation to CRS disease phenotype and severity. Prophage regions were identified using PHASTER. Various in silico tools like ResFinder and VF Analyzer were used to detect virulence genes and antibiotic resistance genes respectively. Progressive MAUVE and maximum likelihood were used for multiple sequence alignment and phylogenetics of prophages respectively. Disease severity of CRS patients was measured using computed tomography Lund–Mackay scores. Fifty-eight S. aureus clinical isolates (CIs) were obtained from 28 CRS patients without nasal polyp (CRSsNP) and 30 CRS patients with nasal polyp (CRSwNP). All CIs carried at least one prophage (average=3.6) and prophages contributed up to 7.7 % of the bacterial genome. Phage integrase genes were found in 55/58 (~95 %) S. aureus strains and 97/211 (~46 %) prophages. Prophages belonging to Sa3int integrase group (phiNM3, JS01, phiN315) (39/97, 40%) and Sa2int (phi2958PVL) (14/97, 14%) were the most prevalent prophages and harboured multiple virulence genes such as sak, scn, chp, lukE/D, sea. Intact prophages were more frequently identified in CRSwNP than in CRSsNP (P=0.0021). Intact prophages belonging to the Sa3int group were more frequent in CRSwNP than in CRSsNP (P=0.0008) and intact phiNM3 were exclusively found in CRSwNP patients (P=0.007). Our results expand the knowledge of prophages in S. aureus isolated from CRS patients and their possible role in disease development. These findings provide a platform for future investigations into potential tripartite associations between bacteria-prophage-human immune system, S. aureus evolution and CRS disease pathophysiology.

Molecular Study of Immune Evasion Cluster Genes in Clinical Isolates of Staphylococcus aureus

Aims: The aim of the present research was to highlight the prevalence of immune evasion cluster genes (IEC) sak, chp, scn, sea, sep among Staphylococcus aureus (S. aureus) clinical isolates. Background: The present study was a cross-sectional retrospective study, included one hundred isolates of S. aureus that were isolated from patients with nosocomial infections. S. aureus isolates were subjected to full microbiological identification and antibiotics sensitivity testing by the disc diffusion method. The presence of IEC genes scn, sea, sak, sep, chpwas determined by polymerase chain reaction (PCR). Methods: The current study included 100 S. aureus isolates; 40% were classified as methicillin resistant. The isolates exhibited marked resistance to beta lactams antibiotics, the lowest resistance was to erythromycin, ciprofloxacin and vancomycin. The presence of one or more IEC was determined in 89 isolates. The prevalence of chp, sak, sea, sep and scn was 54%, 53%, 8%, 7% and 30%, respectively. Results: S. aureus isolates with IEC genes had increased resistance rates to the studied antibiotics; however, this increase was statistically insignificant either to beta-lactam antibiotics, such as amoxacillin/clavulinic acid (P=0.794), ampicillin (P=0.561), cefotaxim (P=0.271), ceftazidime (P=0.145), imipenem (P=0.589) or non beta-lactam antibiotics, such as amikacin (P=0.955) and trimethoprim/sulfamethoxazale (P=0.974). From 40 methicillin resistant Staphylococcus aureus (MRSA) strains, 37 isolates harbor one or more immune evasion cluster genes. Conclusion: The high prevalence of these genes among MRSA may explain its pathogenesis. There is a need for studies with a high number of isolates to verify the present findings.

Genomic Distinctions of LA-MRSA ST398 on Dairy Farms From Different German Federal States With a Low Risk of Severe Human Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) have been found on German dairy farms and may be the cause of difficult-to-treat bovine mastitis. Considering the one health approach, MRSA might be transmitted from animals to humans raising the risk for severe infections. On 17 German dairy farms with a history of MRSA detection, MRSA strains were isolated from quarter milk, bulk tank milk, and swab samples of calves, heifers, pigs, and the environment. A selection of 33 isolates was analyzed using whole-genome sequencing and antimicrobial resistance testing. All detected MRSA strains were attributed to the livestock-associated sequence type 398. Methicillin-resistance was associated with the mecA gene in the staphylococcal cassette chromosome (SCC)mec types IVa (7/33) or V (26/33). The MRSA strains across the German federal states showed large allelic differences indicating independent development and distribution. On one farm, a clonal MRSA isolate was widely spread among different animals and the milking equipment. Moreover, MRSA transmission between two dairy farms in one federal state seems to be likely. In depth studies indicated that the resistance gene prediction and phenotypic resistance are in good agreement. Twenty eight strains were determined to exhibit a non-wildtype phenotype (resistant) against up to seven antimicrobial substances with an overall resistance to β-lactams and tetracycline. Ten different phenotypic antimicrobial resistance patterns were found among the MRSA strains. The strains harbored a wide virulence gene repertoire, of which some of them are related to bovine mastitis. However, the isolates lacked typical human infection associated factors such as the immune evasion cluster genes, staphylococcal enterotoxin genes, or Panton-Valentine leukocidin genes leading to the assumption for a low risk for severe human infections and foodborne diseases.

Prevalence and molecular characterization of MSSA and MRSA among UniSZA undergraduate students in Besut, Terengganu

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is bacteria that resistant to multiple antibiotics, making MRSA infection difficult to treat. Previous studies had indicated that livestock associated MRSA (LA-MRSA) is presence in livestock and able to transmit to human that have close contacts with animals. In the past, LA-MRSA is always related with the presence of tetracycline resistance and lack of immune evasion cluster (IEC) while MRSA strain originated from human posses IEC but without tetracycline resistant genes. LA-MRSAs with IEC genes may able to infect human more efficiently as the human innate immunity cannot respond to the LA-MRSA invasion. This study was carried out to study the prevalence of methicillin-susceptible S. aureus (MSSA) and MRSA among UniSZA students that have previous exposure to animal as well to detect the presence of IEC and tetracycline resistance genes in the MSSA isolates. Methods and materials: A total of 400 swab samples (200 oral and nasal respectively) were collect from 200 students that belongs to 5 different undergraduate courses in UniSZA. Isolation of S. aureus was done using phenotypic identification methods, followed by detection of nuc and mecA genes using PCR. Isolates that been identified as MSSA were later tested using PCR to determine the presence of IEC and tetracycline resistance genes. The antibiotic susceptibility pattern of MSSA isolates were also been investigated. Results: 87 isolates are confirmed to contain nuc gene by using PCR and no mecA gene were detected, indicating the absent of MRSA from students. MSSA isolates show resistances towards various antibiotics (chlorampenicol, oxacillin, amikacin, tetracycline and cephalothin), suggesting that the emergence of antimicrobial resistance MSSA among the students. 3.1% (2/65) isolates shown the presence of IEC type E while 43 (66.2%; 43/65) MSSA contain only sak genes. No tetracyclines resistance genes were detected. Conclusion: As the MSSA isolated from these students may already spread among the animals, antibiotic awareness, careful monitoring and responsible usage of antibiotics must be carried out to avoid the emergence of multiple drugs resistant MSSA to be transmitted between species.

Complete genome of a methicillin-resistant Staphylococcus vitulinus from Danish ground beef meat carrying a mecA2 resistance gene and a novel ccr allotype

ObjectivesTo report the complete genome sequence of a methicillin-resistant Staphylococcus vitulinus from ground beef to allow comparison with other available S. vitulinus genomes and to investigate its SCCmec element. MethodsMeat samples from grocery stores in Denmark were examined for the presence of staphylococcal species by plating on selective plates. One colony isolated from beef was identified as S. vitulinus by MALDI-TOF and genome sequenced using a combination of Illumina and Oxford Nanopore technologies. Phylogenetic and in silico resistome analyses were performed for all available S. vitulinus genomes. ResultsThe closed genome of S. vitulinus Tienloo1 isolate had a chromosome size of 2,628,028 bp and contained a single novel 2,380 bp plasmid based on a hybrid assembly. It carried mecA as the only resistance marker. The isolate was found not to carry any immune evasion cluster genes, which have been putatively associated to human origin. Comparison with all publicly available S. vitulinus draft genomes showed a diverse population and revealed that only the Danish beef isolate contained a mec gene in addition to a ccr gene complex. Additionally, the single ccrC gene within the isolate was novel and distant from the mecA2 gene. ConclusionThis isolate, Tienloo1, from a ground beef meat sample represents the first complete genome of S. vitulinus found to carry a mecA2 gene and a novel ccr allotype in its SCCmec element that is distinct from all publicly available draft S. vitulinus genomes.

Determination of antibiotic resistance genes, immune evasion cluster and agr types among Staphylococcus aureus strains isolated from children with adenoiditis

Adenoiditis is the most common infection in childhood and different microorganism such as viruses and bacteria cause this infection. Staphylococcus aureus (S. aureus) is the main Gram-positive bacterium that can involve in adenoiditis. The aim of this study was to determination of antibiotic resistance, immune evasion cluster genes and agr types among S. aureus isolates were collected from children with adenoiditis. Totally 36 clinical isolates of S. aureus were obtained from 112 children suffering from adenoiditis. Susceptibility of the isolates to different antimicrobial agents were determined using standard disk diffusion method. PCR technique was used for detection of resistance and virulence genes. SCCmec and agr typing were used for molecular typing of the isolates. All isolates were sensitive to vancomycin and 47.2% of isolates were resistant to penicillin. Twenty-two present of isolates were considered as MRSA and SCCmec type IV was detected in 5 MRSA isolates. Only, agr types I and II were identified among the isolates. ermC was the predominant macrolide resistance gene and ant(4′)-Ia was the most common aminoglycoside resistance gene. Virulence genes including sak, chp and sea were identified in 47.2% (n = 17), 30.5% (n = 11) and 13.8% (n = 5). This study was the first report in Iran, Kerman, about determination of antibiotic resistance, virulence genes, agr groups and SCCmec types among the clinical S. aureus isolates were collected from children with adenoiditis. Also, our results can be helpful in empirical therapy and increase our knowledge about genetic characteristics of S. aureus isolates are involved to adenoiditis.

Livestock-associated methicillin-resistant Staphylococcus aureus: Establishing links between animals and humans on livestock holdings.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) represents a concern in both human and veterinary medicine. The aim of this study was to investigate potential LA-MRSA transmission between animals and humans in rural settings. To this aim, a study was designed to include 14 farms in Slovenia, which were selected on the basis of a farmer (initial patient) with confirmed LA-MRSA infection and regular animal contacts. On all farms, the initial patients, their household members, animals and barn environment were analysed for the presence of LA-MRSA. In addition, the epidemiologically linked hospital-related LA-MRSA isolates were included to investigate possible nosocomial transmissions. On five farms, LA-MRSA was discovered both in animals and in humans. In total, 49 LA-MRSA isolates of different origins underwent whole-genome sequencing, antimicrobial susceptibility testing and spa typing. All 49 isolates belonged to the sequence type 398 (ST398), spa types t011 and t034, and harboured staphylococcal chromosomal cassette mec Vc. High levels of concordance between resistance phenotypes and genotypes were observed. No transmission pairs between animals and initial patients were discovered. However, several isolates originating from farm animals and other household members formed clusters with pairwise distances of ≤14 single nucleotide polymorphisms (SNPs), indicating recent transmission events. In addition, three closely related isolates (0 SNP) form hospitalized patients were observed, indicating a possible nosocomial transmission. Two hospital-related isolates harboured the immune evasion cluster genes, which are associated with adaptation to the human host; however, these two isolates differed in >30 SNPs from the remaining isolates. Characteristics of LA-MRSA from Slovenia reflect those observed previously in other European studies. Immune evasion cluster-positive LA-MRSA ST398 suggests its re-adaptation to the human host and calls for a closer monitoring of such emerging LA-MRSA lineages, in addition to monitoring and preventing the introduction of LA-MRSA from farms to hospitals where transmission is highly plausible.

Simultaneous Nasal Carriage by Methicillin-Resistant and Methicillin Susceptible Staphylococcus aureus of Lineage ST398 in a Live Pig Transporter.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST)398 is a livestock associated (LA) lineage with zoonotic potential, especially in humans with live pig contact. The objective of this study was to characterize two S. aureus strains of lineage ST398 (one methicillin-resistant (MRSA), one methicillin-susceptible (MSSA)) isolated from the same nasal sample of a patient admitted in the Intensive-Care Unit of a Spanish Hospital, and with previous occupational exposure to live pigs, by whole-genome-sequencing (WGS). The sample was obtained during routine surveillance for MRSA colonization. Purified genomic DNA was sequenced using Illumina HiSeq 2000 and processed using conventional bioinformatics software. The two isolates recovered were both S. aureus t011/ST398 and showed similar resistance-phenotypes, other than methicillin susceptibility. The possession of antibiotic resistance genes was the same, except for the mecA-gene located in SCCmecV in the MRSA isolate. The MSSA isolate harbored remnants of a SCCmec following the deletion of 17342bp from a recombination between two putative primases. Both isolates belonged to the livestock-associated clade as defined by three canonical single-nucleotide-polymorphisms, and neither possessed the human immune evasion cluster genes, chp, scn, or sak. The core genome alignment showed a similarity of 99.6%, and both isolates harbored the same mobile genetic elements. The two nasal ST398 isolates recovered from the patient with previous occupational exposure to pigs appeared to have a livestock origin and could represent different evolutionary steps of animal-human interface lineage. The MSSA strain was formed as a result of the loss of the mecA gene from the livestock-associated-MRSA lineage.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) in Purulent Subcutaneous Lesions of Farm Rabbits.

Methicillin-resistant Staphylococcus aureus (MRSA) are one of the main pathogens associated with purulent infections. MRSA clonal complex 97 (CC97) has been identified in a wide diversity of livestock animals. Therefore, we aimed to investigate the antibiotic resistance profiles of MRSA strains isolated from purulent lesions of food-producing rabbits. Samples from purulent lesions of 66 rabbits were collected in a slaughterhouse in Portugal. Samples were seeded onto ORSAB plates with 2 mg/L of oxacillin for MRSA isolation. Susceptibility to antibiotics was tested by the disk diffusion method against 14 antimicrobial agents. The presence of resistance genes, virulence factors and the immune evasion cluster (IEC) system was studied by polymerase chain reaction. All isolates were characterized by multilocus sequence typing (MLST), agr and spa typing. From the 66 samples analyzed, 16 (24.2%) MRSA were detected. All strains were classified as multidrug-resistant as they were resistant to at least three classes of antibiotics. All isolates showed resistance to penicillin, erythromycin and clindamycin. Seven isolates were resistant to gentamicin and harbored the aac(6′)-Ie-aph (2″)-Ia gene. Resistance to tetracycline was detected in 10 isolates harboring the tet(K) gene. The IEC genes were detected in three isolates. MRSA strains belonged to CC97, CC1, CC5, CC15 or CC22. The isolates were assigned to six different spa types. In this study we found a moderate prevalence of multidrug-resistant MRSA strains in food-producing rabbits. This may represent concern for food safety and public health, since cross-contamination may occur, leading to the spread of MRSA and, eventually, the possibility of ingestion of contaminated meat.

Differences in virulence between the two more prevalent Staphylococcus aureus clonal complexes in rabbitries (CC121 and CC96) using an experimental model of mammary gland infection

Staphylococcal mastitis is a major health problem in humans and livestock that leads to economic loss running in millions. This process is currently one of the main reasons for culling adult rabbit does. Surprisingly, the two most prevalent S. aureus lineages isolated from non-differentiable natural clinical mastitis in rabbits (ST121 and ST96) generate different immune responses. This study aimed to genetically compare both types of strains to search for possible dissimilarities to explain differences in immune response, and to check whether they showed similar virulence in in vitro tests as in experimental intramammary in vivo infection. The main differences were observed in the enterotoxin gene cluster (egc) and the immune-evasion-cluster (IEC) genes. While isolate ST121 harboured all six egc cluster members (seg, sei, selm, seln, selo, selu), isolate ST96 lacked the egc cluster. Strain ST96 carried a phage integrase Sa3 (Sa3int), compatible with a phage integrated into the hlb gene (β-haemolysin-converting bacteriophages) with IEC type F, while isolate ST121 lacked IEC genes and the hlb gene was intact. Moreover, the in vitro tests confirmed a different virulence capacity between strains as ST121 showed greater cytotoxicity for erythrocytes, polymorphonuclear leukocytes and macrophages than strain ST96. Differences were also found 7 days after experimental intramammary infection with 100 colony-forming units. The animals inoculated with strain ST121 developed more severe gross and histological mastitis, higher counts of macrophages in tissue and of all the cell populations in peripheral blood, and a significantly larger total number of bacteria than those infected by strain ST96.

Molecular Epidemiology of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in Wild, Captive and Laboratory Rats: Effect of Habitat on the Nasal S. aureus Population.

Rats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Free-living wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S. aureus lineages—many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S. aureus, respectively.

Methicillin-Resistant Staphylococcus aureus CC398 in Purulent Lesions of Piglets and Fattening Pigs in Portugal.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is an important clinical problem. In 2005, a livestock-associated MRSA clone was described, named CC398, being mostly associated with pigs, and causing colonization and infection in pigs and in related humans. The prevalence of these strains in food-producing pigs raised concerns about the possibility of MRSA-CC398 being a foodborne pathogen. The objective of this study was to investigate the presence of S. aureus and MRSA in 141 carcasses of pigs at three slaughterhouses of Portugal, discarded from the food chain by signs of infection, and to characterize the recovered isolates. Methods: S. aureus isolates were identified by matrix-assisted laser desorption/ionization time-of-flight and they were typed (spa, CC398-clone, and SCCmec). The study of antibiotic resistance and virulence genes, and the detection of immune evasion cluster genes and prophages were performed by PCR and sequencing. Results: Twenty-eight S. aureus were obtained from 141 samples (one/sample, 19.9%), being 22 MRSA and 6 methicillin-susceptible S. aureus (MSSA). All MRSA strains were typed as CC398 and were ascribed to three spa types (t011, t108, and t1451). The SCCmec detected differed according to the spa types of MRSA isolates (SCCmecV: t011 and t108; SCCmecIVa: t1451). The MSSA strains were classified as spa-t1491-ST1-CC1. All the strains contained a wide range of antimicrobial resistance genes, the resistance to tetracycline being the prevalent one. In contrast, the strains contained only a few virulence genes. Among the 6 integrases of phages tested, three were detected: SΦ1, SΦ2, and SΦ7, with variations between MRSA and MSSA strains. Conclusions: MRSA-CC398 is not only a habitual pig colonizer but also an opportunistic pathogen in these animals, and must be controlled at the level of producers and slaughterhouses because of its impact on public health.

Nasal Colonization of Humans with Occupational Exposure to Raw Meat and to Raw Meat Products with Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is widely disseminated as a nasal colonizer of conventionally raised livestock and of humans subjected to occupational exposure. Reports on contamination of raw meat raise the question as to whether occupationally exposed food handlers are at particular risk of nasal colonization by LA-MRSA. Here, we report the results from a cross-sectional study on nasal S. aureus/MRSA colonization of butchers, meat sellers, and cooks in Germany. We sampled 286 butchers and meat sellers in 26 butcheries and 319 cooks handling meat in 16 professional canteen kitchens. Swabs were processed on both blood agar plates and MRSA-selective plates. MRSA were confirmed by PCR for mec genes and by broth microdilution. All isolates were subjected to molecular typing. PCR for markers useful to differentiate human-adapted and animal-adapted subpopulations was performed due to the presence of clonal complexes known to occur in both livestock and humans (CC5, CC7, CC8, CC9, and CC398). Only two participants (0.33%) were colonized by MRSA (Hospital-associated MRSA ST22). Nasal colonization by methicillin-susceptible S. aureus (MSSA) was detected in 16.6% of cooks and in 26.2% of butchers and meat sellers. Among 16 of the isolates attributed to CC7, three were negative for the immune evasion gene cluster, suggesting an animal origin. Isolates attributed to CC5, CC8, and CC398 were negative for markers typical of animal-adapted subpopulations. The occupational handling of raw meat and raw meat products was not associated with nasal colonization by LA-MRSA.

Phylogenomic Classification and the Evolution of Clonal Complex 5 Methicillin-Resistant Staphylococcus aureus in the Western Hemisphere.

Clonal complex 5 methicillin-resistant Staphylococcus aureus (CC5-MRSA) includes multiple prevalent clones that cause hospital-associated infections in the Western Hemisphere. Here, we present a phylogenomic study of these MRSA to reveal their phylogeny, spatial and temporal population structure, and the evolution of selected traits. We studied 598 genome sequences, including 409 newly generated sequences, from 11 countries in Central, North, and South America, and references from Asia and Europe. An early-branching CC5-Basal clade is well-dispersed geographically, is methicillin-susceptible and MRSA predominantly of ST5-IV such as the USA800 clone, and includes separate subclades for avian and porcine strains. In the early 1970s and early 1960s, respectively, two clades appeared that subsequently underwent major expansions in the Western Hemisphere: a CC5-I clade in South America and a CC5-II clade largely in Central and North America. The CC5-I clade includes the ST5-I Chilean/Cordobes clone, and the ST228-I South German clone as an early offshoot, but is distinct from other ST5-I clones from Europe that nest within CC5-Basal. The CC5-II clade includes divergent strains of the ST5-II USA100 clone, various other clones, and most known vancomycin-resistant strains of S. aureus, but is distinct from ST5-II strain N315 from Japan that nests within CC5-Basal. The recombination rate of CC5 was much lower than has been reported for other S. aureus genetic backgrounds, which indicates that recurrence of vancomycin resistance in CC5 is not likely due to an enhanced promiscuity. An increased number of antibiotic resistances and decreased number of toxins with distance from the CC5 tree root were observed. Of note, the expansions of the CC5-I and CC5-II clades in the Western Hemisphere were preceded by convergent gains of resistance to fluoroquinolone, macrolide, and lincosamide antibiotics, and convergent losses of the staphylococcal enterotoxin p (sep) gene from the immune evasion gene cluster of phage ϕSa3. Unique losses of surface proteins were also noted for these two clades. In summary, our study has determined the relationships of different clades and clones of CC5 and has revealed genomic changes for increased antibiotic resistance and decreased virulence associated with the expansions of these MRSA in the Western Hemisphere.