Whole-Genome Sequence of Livestock-Associated ST398 Methicillin-Resistant Staphylococcus aureus Isolated from Humans in Canada

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.     

Detection of Methicillin-Susceptible Staphylococcus aureus ST398 and ST133 Strains in Gut Microbiota of Healthy Humans in Spain

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n?=?11) and sequences types (n?=?8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4′)-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.     

Survival of Staphylococcus aureus ST398 in the Human Nose after Artificial Inoculation

There is evidence that MRSA ST398 of animal origin is only capable of temporarily occupying the human nose, and it is therefore, often considered a poor human colonizer.We inoculated 16 healthy human volunteers with a mixture of the human MSSA strain 1036 (ST931, CC8) and the bovine MSSA strain 5062 (ST398, CC398), 7 weeks after a treatment with mupirocin and chlorhexidine-containing soap. Bacterial survival was studied by follow-up cultures over 21 days. The human strain 1036 was eliminated faster (median 14 days; range 2–21 days) than the bovine strain 5062 (median 21 days; range 7–21 days) but this difference was not significant (p = 0.065). The bacterial loads were significantly higher for the bovine strain on day 7 and day 21. 4/14 volunteers (28.6%) showed elimination of both strains within 21 days. Of the 10 remaining volunteers, 5 showed no differences in bacterial counts between both strains, and in the other 5 the ST398 strain far outnumbered the human S. aureus strain. Within the 21 days of follow-up, neither human strain 1036 nor bovine strain 5062 appeared to acquire or lose any mobile genetic elements. In conclusion, S. aureus ST398 strain 5062 is capable of adequately competing for a niche with a human strain and survives in the human nose for at least 21 days.     

Colonization and transmission of methicillin-resistant Staphylococcus aureus ST398 in nursery piglets

A transmission experiment was performed to evaluate the spread of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in nursery piglets. Reproduction ratios (R(0)) in three experimental groups were found to vary between 3.92 and 52.54, indicating that after introduction, MRSA ST398 will spread easily among weaned piglets, with a tendency to become established.     

Proteome of a methicillin-resistant Staphylococcus aureus clinical strain of sequence type ST398

Proteomics is a powerful tool to analyze the differences in gene expression of bacterial strains. Staphylococcus aureus has long been recognized as an important pathogen in human disease. In order to investigate this pathogen, the proteome of a clinical methicillin-resistant S. aureus (MRSA) strain of the sequence type ST398 was determined using 2-DE. Using 2-DE we obtained a total of 105 spots the MRSA strain. Furthermore in correlation with bioinformatic databases, they allowed accurate identification and characterization of proteins, resulting in 227 identified proteins. There were found proteins related to basic function of the cell, but also proteins related to virulence like catalase, specific of S. aureus species, and proteins related to antibiotic resistance. Proteins associated with antibiotic resistance or virulence factors are related to genomic databases. The most abundant classes identified involved glycolysis, energy production, one-carbon metabolism, and oxidation-reduction process, all of which reflect an active metabolism. These results highlight the importance of proteomics to deepen in the knowledge of protein expression of MRSA strain of the lineage ST398, microorganism with diverse and important resistance mechanisms. With this proteome map we have an essential tool for a better understanding of this pathogen and providing new data for protein databases. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.     

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation

Aims: Methicillin‐resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. Methods and results: Twelve specific pathogen‐free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10⁴ colony‐forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. Conclusion: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. Significance and Impact of the Study: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10⁴ colony‐forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.     

Staphylococcus aureus,Including Meticillin-Resistant Staphylococcus aureus,among General Practitioners and Their Patients: A Cross-Sectional Study

BackgroundThe role of general practitioners (GPs) as reservoir and potential source for Staphylococcus aureus (SA) transmission is unknown. Our primary objective was to evaluate the prevalence of SA and community-acquired methicillin resistant SA (CA-MRSA) carrier status (including spa typing) among GPs and their patients in Belgium. The secondary objective was to determine the association between SA/CA-MRSA carriage in patients and their characteristics, SA carriage in GPs, GP and practice characteristics.MethodsThe Belgian GPs, who swabbed their patients in the APRES study (which assessed the prevalence of SA nasal carriage in nine European countries; November 2010 –June 2011), were asked to swab themselves as well (May-June 2011). GPs and their patients had to complete a questionnaire on factors related to SA carriage and transmission. SA isolation including CA-MRSA and spa typing was performed on the swabs.ResultsIn eighteen practices 34 GPs swabbed patients of which 25 GPs provided personal swabs. The analysis was performed on 3008 patient records. Among GPs SA carriage (28%) was more prevalent than among their patients (19.2%), but CA-MRSA carriage was not present. SA was more prevalent among younger patients and those living with cattle. Spa typing SA and MRSA strains did not suggest correlation within practices or between patients and GPs, but chronic skin conditions of GPs and always handshaking patients by SA positive GPs were associated with more SA among patients, and hand washing after every patient contact with less SA among patients in practices with high antibiotic prescribing rates.ConclusionNo MRSA was found among GPs, although their SA carriership was higher compared to their patients’. Spa types did not cluster within practices, possibly due to difference in timing of swabbing. To minimise SA transmission to their patients GPs should consider taking appropriate care of their chronic skin diseases, antibiotic prescribing behaviour, handshaking and hand washing habits.     

Nasal Colonization of Humans with Methicillin-Resistant Staphylococcus aureus (MRSA) CC398 with and without Exposure to Pigs

Background

Studies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure to colonized animals. The study presented here addresses the question of further transmission to non exposed humans.

Methods

After selecting 47 farms with colonized pigs in different regions of Germany we sampled the nares of 113 humans working daily with pigs and of their 116 non exposed family members. The same was performed in 18 veterinarians attending pig farms and in 44 of their non exposed family members. For investigating transmission beyond families we samples the nares of 462 pupils attending a secondary school in a high density pig farming area. MRSA were detected by direct culture on selective agar. The isolates were typed by means of spa-sequence typing and classification of SCCmec elements. For attribution of spa sequence types to clonal lineages as defined by multi locus sequence typing we used the BURP algorithm. Antibiotic susceptibility testing was performed by microbroth dilution assay.

Results

At the farms investigated 86% of humans exposed and only 4.3% of their family members were found to carry MRSA exhibiting spa-types corresponding to clonal complex CC398. Nasal colonization was also found in 45% of veterinarians caring for pig farms and in 9% of their non exposed family members. Multivariate analysis revealed that antibiotic usage prior to sampling beard no risk with respect to colonization. From 462 pupils only 3 were found colonized, all 3 were living on pig farms.

Conclusion

These results indicate that so far the dissemination of MRSA CC398 to non exposed humans is infrequent and probably does not reach beyond familial communities.     

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.     

The Emergence and Spread of Multiple Livestock-Associated Clonal Complex 398 Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains among Animals and Humans in the Republic of Ireland, 2010–2014

Clonal complex (CC) 398 methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) are associated with carriage and infection among animals and humans but only a single case of CC398 MRSA has been reported in the Republic of Ireland (ROI). The present study investigated the molecular epidemiology of CC398 MRSA (n = 22) and MSSA (n = 10) from animals and humans in the ROI from 2010–2014. Isolates underwent antimicrobial susceptibility testing, spa typing, DNA microarray profiling and PCR for CC398-associated resistance genes. All MRSA underwent SCCmec IV or V subtyping. Four distinct CC398-MRSA incidents were identified from (i) a man in a nursing home (spa type t011-SCCmec IVa, immune evasion complex (IEC) negative), (ii) a horse and veterinarian who had recently travelled to Belgium (t011-IVa, IEC positive), (iii) pigs (n = 9) and farm workers (n = 9) on two farms, one which had been restocked with German gilts and the other which was a finisher farm (t034-V_T, IEC negative, 3/9 pigs; t011- V_T, IEC negative, 6/9 pigs & 9/9 farm workers), and (iv) a child who had worked on a pig farm in the UK (t034-V_T, IEC negative). Isolates also carried different combinations of multiple resistance genes including erm(A), erm(B), tet(K), tet(M) & tet(L), fexA, spc, dfrG, dfrK aacA-aphD and aadD further highlighting the presence of multiple CC398-MRSA strains. CC398 MSSA were recovered from pigs (n = 8) and humans (n = 2). CC398 MSSA transmission was identified among pigs but zoonotic transmission was not detected with animal and human isolates exhibiting clade-specific traits. This study highlights the importation and zoonotic spread of CC398 MRSA in the ROI and the spread of CC398 MSSA among pigs. Increased surveillance is warranted to prevent further CC398 MRSA importation and spread in a country that was considered CC398 MRSA free.     

Clonal Dynamics of Nasal Staphylococcus aureus and Staphylococcus pseudintermedius in Dog-Owning Household Members. Detection of MSSA ST398

The objective of this study was to investigate the dynamics of nasal carriage by Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP) among healthy dog-owning household members involved in 7 previous index cases of suspected anthropozoonotic (n = 4) and zoonotic (n = 3) interspecies transmission [4 direct cases, identical SA (n = 3) or SP (n = 1) in owner and dog; three indirect, SP in owner (n = 2) or SA in dog (n = 1)]. Co-carriage with methicillin-resistant coagulase-negative staphylococci (MRCoNS) was also evaluated. Sixteen owners and 10 dogs were sampled once every three months for one year. In total, 50 SA and 31 SP were analysed by MLST, and SA also by spa typing. All isolates were subjected to ApaI/SmaI-PFGE and antimicrobial resistance and virulence profiles were determined. All index owners were persistent SA carriers in all direct-anthropozoonotic transmission cases, while only one dog was persistent SA carrier. Owner and dog exhibited a persistent SP carriage status in the direct-zoonotic transmission case. SP was maintained in the index human over time in one indirect-zoonotic transmission case. Only one SP was methicillin-resistant. SA belonged to genetic backgrounds of MRSA pandemic clones: CC45, CC121, CC30, CC5 and CC398. Three individuals carried a MSSA t1451-ST398 clone with the erm(T)-cadD/cadX resistance genes. SA or SP were persistently detected in the nasal cavity of 7 (43.8%) and 2 (12.5%) owners, and in one and 2 dogs, respectively. SA was recovered as the single species in 10 owners and in one dog; SP in 3 owners and 4 dogs; and both bacterial species in one owner and 4 dogs. Co-carriage of SA or SP with MRCoNS isolates was common (30.7%). This is the first study on the dynamics of nasal carriage of SA and SP in healthy pet-owning household members. Dog-contact may play a role in the staphylococcal species distribution of in-contact individuals.     

The emerging methicillin-resistant Staphylococcus aureus ST398 clone can easily be typed using the Cfr9I SmaI-neoschizomer

Aim:  To establish a PFGE protocol using Cfr9I, neoschizomer of SmaI, for typing of Staphylococcus aureus isolates belonging to the emerging MRSA ST398 clone.
Methods and Results:  Staphylococcus aureus ST398 and non-ST398 isolates were analysed using the PFGE conditions recommended by the HARMONY consensus protocol. Genomic DNA of non-ST398 isolates could be digested with SmaI, XmaI (also a SmaI-neoschizomer) and Cfr9I. The DNA of SmaI-nontypeable ST398 isolates was partially resistant to XmaI, but could be digested with Cfr9I. By PCR-amplification/sequencing, the presence of a novel C5-cytosine methyltransferase gene ( sauST398M ) was detected in the ST398 isolates. The encoded enzyme, which shows high similarity with C5-cytosine methyltransferases that modify the CCCGGG recognition sequence, could be responsible for the different restriction results.
Conclusion:  SmaI-PFGE is regarded as the 'gold standard' for typing S. aureus . Because of different susceptibility of the GGGCCC recognition sites of the ST398 DNA against SmaI, XmaI and Cfr9I, the proposed protocol is a valuable tool for ST398 typing.
Significance and Impact of the Study:  The use of this protocol allows the comparison of results from SmaI-nontypeable isolates with S. aureus SmaI-PFGE databases and can be applied for outbreak investigations and traceability studies of this emerging MRSA clone.     

Methicillin-resistant Staphylococcus aureus ST9 in pigs in Thailand

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial and community-associated pathogen. Recently, livestock-associated MRSA (LA-MRSA) has emerged and disseminated in Europe and North America and now constitutes a considerable zoonotic burden in humans with risk factors of pig exposure, whereas the extent of the livestock reservoir is relatively unknown on other continents.

Methodology/Principal Findings

From March through April 2011, MRSA was identified in pigs from 3 out of 30 production holdings in Chang Mai Province, Thailand. Representative isolates were subjected to molecular characterization and antimicrobial susceptibility testing; all isolates had genotypic and phenotypic characteristics of LA-MRSA previously characterized in the region: they belonged to ST9, lacked the lukF-lukS genes encoding Panton-Valentine leukocidin, and were resistant to multiple non-β-lactam antimicrobials. However, unlike other Asian LA-MRSA-ST9 variants, they were spa type t337 and harbored a different staphylococcal cassette chromosome mec IX.

Conclusions/Significance

A novel MRSA-ST9 lineage has been established in the pig population of Thailand, which differs substantially from LA-MRSA lineages found in other areas of the continent. The emergence of novel LA-MRSA lineages in the animal agriculture setting is worrisome and poses a serious threat to global public health.     

Methicillin-Resistant Staphylococcus aureus (MRSA) Strain ST398 Is Present in Midwestern U.S. Swine and Swine Workers

Background

Recent research has demonstrated that many swine and swine farmers in the Netherlands and Canada are colonized with MRSA. However, no studies to date have investigated carriage of MRSA among swine and swine farmers in the United States (U.S.).

Methods

We sampled the nares of 299 swine and 20 workers from two different production systems in Iowa and Illinois, comprising approximately 87,000 live animals. MRSA isolates were typed by pulsed field gel electrophoresis (PFGE) using SmaI and EagI restriction enzymes, and by multi locus sequence typing (MLST). PCR was used to determine SCCmec type and presence of the pvl gene.

Results

In this pilot study, overall MRSA prevalence in swine was 49% (147/299) and 45% (9/20) in workers. The prevalence of MRSA carriage among production system A''s swine varied by age, ranging from 36% (11/30) in adult swine to 100% (60/60) of animals aged 9 and 12 weeks. The prevalence among production system A''s workers was 64% (9/14). MRSA was not isolated from production system B''s swine or workers. Isolates examined were not typeable by PFGE when SmaI was used, but digestion with EagI revealed that the isolates were clonal and were not related to common human types in Iowa (USA100, USA300, and USA400). MLST documented that the isolates were ST398.

Conclusions

These results show that colonization of swine by MRSA was very common on one swine production system in the midwestern U.S., suggesting that agricultural animals could become an important reservoir for this bacterium. MRSA strain ST398 was the only strain documented on this farm. Further studies are examining carriage rates on additional farms.     

Recent Emergence of Staphylococcus aureus Clonal Complex 398 in Human Blood Cultures

Background

Recently, a clone of MRSA with clonal complex 398 (CC398) has emerged that is related to an extensive reservoir in animals, especially pigs and veal calves. It has been reported previously that methicillin-susceptible variants of CC398 circulate among humans at low frequency, and these have been isolated in a few cases of bloodstream infections (BSI). The purpose of this study was to determine the prevalence of S. aureus CC398 in blood cultures taken from patients in a geographic area with a high density of pigs.

Methodology/Principal Findings

In total, 612 consecutive episodes of S. aureus BSI diagnosed before and during the emergence of CC398 were included. Three strains (2 MSSA and 1 MRSA) that were isolated from bacteremic patients between 2010–2011 were positive in a CC398 specific PCR. There was a marked increase in prevalence of S. aureus CC398 BSI isolated between 2010–2011 compared to the combined collections that were isolated between 1996–1998 and 2002–2005 (3/157, 1.9% vs. 0/455, 0.0%; p = 0.017).

Conclusions/Significance

In conclusion, in an area with a relative high density of pigs, S. aureus CC398 was found as a cause of BSI in humans only recently. This indicates that S. aureus CC398 is able to cause invasive infections in humans and that the prevalence is rising. Careful monitoring of the evolution and epidemiology of S. aureus CC398 in animals and humans is therefore important.     

Transmission of Methicillin-Resistant Staphylococcus aureus CC398 from Livestock Veterinarians to Their Household Members

There are indications that livestock-associated MRSA CC398 has a reduced human-to-human transmissibility, limiting its impact on public health and justifying modified control measures. This study determined the transmissibility of MRSA CC398 from livestock veterinarians to their household members in the community as compared to MRSA non-CC398 strains. A one-year prospective cohort study was performed to determine the presence of MRSA CC398 in four-monthly nasal and oropharyngeal samples of livestock veterinarians (n  =  137) and their household members (n  =  389). In addition, a cross-sectional survey was performed to detect the presence of MRSA non-CC398 in hospital derived control patients (n  =  20) and their household members (n  =  41). Staphylococcus aureus isolates were genotyped by staphylococcal protein A (spa) typing and multiple-locus variable-number tandem repeat analysis (MLVA). Mean MRSA CC398 prevalence over the study period was 44% (range 41.6–46.0%) in veterinarians and 4.0% (range 2.8–4.7%) in their household members. The MRSA CC398 prevalence in household members of veterinarians was significantly lower than the MRSA non-CC398 prevalence in household members of control patients (PRR 6.0; 95% CI 2.4–15.5), indicating the reduced transmissibility of MRSA CC398. The impact of MRSA CC398 appears to be low at the moment. However, careful monitoring of the human-to-human transmissibility of MRSA CC398 remains important.