Interactions of Methicillin Resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in Polymicrobial Wound Infection

May combine two claims.

Staining with epidermal marker, K14 specific antibody, confirmed delayed epithelialization in infected wounds when compared to non-infected control wounds, with more pronounced inhibition of wound closure in mixed-species infected wounds.

“Staining with epidermal marker, K14 specific antibody, confirmed delayed epithelialization in infected wounds when compared to non-infected control wounds ( Fig. 4B ), with more pronounced inhibition of wound closure in mixed-species infected wounds.”

May combine two claims.

Significant suppression of KGF1 was found in wounds infected by mixed-species biofilms, while single species infection did not result in suppression of KGF1 expression.

“Significant suppression of KGF1 was found in wounds infected by mixed-species biofilms ( Fig. 4A ), while single species infection did not result in suppression of KGF1 expression.”

May combine two claims.

Both USA300 and P. aeruginosa infected wounds showed delayed epithelialization when compared to control, uninfected wounds, however statistically significant differences were seen only in USA300 infected wounds.

“Both USA300 and P. aeruginosa infected wounds showed delayed epithelialization when compared to control, uninfected wounds, however statistically significant differences were seen only in USA300 infected wounds ( Fig. 3B ).”

May combine two claims.

USA300 and P. aeruginosa co-existed in the wounds; and the growth of USA300 was slightly reduced by P. aeruginosa 09-010 in vivo, by 1.5 and 1.6 log on days 2 and 4 after wounding, respectively.

“USA300 and P. aeruginosa co-existed in the wounds; and that the growth of USA300 was slightly reduced by P. aeruginosa 09-010 in vivo, by 1.5 and 1.6 log on days 2 and 4 after wounding, respectively ( Fig. 2B ).”

May combine two claims.

Genes encoding autoinducer synthesis protein RhlI (RhlI) and a key enzyme in alginate production, GDP-mannose 6-dehydrogenase (AlgD), were present in all strains tested.

“Genes encoding autoinducer synthesis protein RhlI (RhlI) and a key enzyme in alginate production, GDP-mannose 6-dehydrogenase (AlgD), were present in all strains tested.”

May combine two claims.

Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo.

“Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo.”

• Also stated in: § P. aeruginosa 09-010 Suppresses the Growth of USA300 in vitro and Less Efficiently in vivo · plain

  “In summary, Pseudomonas has shown an inhibitory effect on USA300 growth in the mixed-species biofilms in vitro, while this effect was not as profound in vivo.”

May combine two claims.

Only one of the strains tested, P. aeruginosa 09-010, had a gene encoding ExoS, while the isolates 09-007 and 09-008 did not contain ToxA nor autoinducer synthesis protein LasI.

“Only one of the strains tested, P. aeruginosa 09-010, had a gene encoding ExoS, while the isolates 09-007 and 09-008 did not contain ToxA nor autoinducer synthesis protein LasI ( Fig. 1A ).”

May combine two claims.

There was no cross-contamination between the wounds as wounds inoculated with USA300 showed no detectable Pseudomonas, and no USA300 was recovered in P. aeruginosa 09-010 inoculated wounds.

“There was no cross-contamination between the wounds as wounds inoculated with USA300 showed no detectable Pseudomonas, and no USA300 was recovered in P. aeruginosa 09-010 inoculated wounds.”

May combine two claims.

The average rate of epithelialization at day 2 for USA300, P. aeruginosa, and wounds infected with both species was 42%, 57%, and 35%, respectively, as compared to 74% wound closure in control uninfected wounds.

“The average rate of epithelialization at day 2 for USA300, P. aeruginosa, and wounds infected with both species was 42%, 57%, and 35%, respectively, as compared to 74% wound closure in control uninfected wounds ( Fig. 3A ).”

May combine two claims.

Although bacterial counts were still significantly high at day 4 post-wounding, the expression of all tested cytokines was similar to expression levels detected in normal unwounded skin.

“Although bacterial counts were still significantly high at day 4 post-wounding ( Fig. 2 ) the expression of all tested cytokines was similar to expression levels detected in normal unwounded skin (data not shown).”

May combine two claims.

In the porcine model, the wound environment allowed coexistence of both species and the growth of USA300 was only slightly reduced in comparison to the reduction observed in vitro.

“Despite the fact that P. aeruginosa inhibited the growth of USA300 in vitro similarly to effects previously reported in murine and rabbit wound polymicrobial infection [42], in the porcine model, the wound environment allowed coexistence of both species and the growth of USA300 was only slightly reduced in comparison to the reduction observed in vitro ( Fig. 2 ).”

May combine two claims.

IL-6 also showed an increasing trend in USA300 infected wounds; however these changes were not significant in comparison to control, non-inoculated wounds.

“IL-6 also showed an increasing trend in USA300 infected wounds; however these changes were not significant in comparison to control, non-inoculated wounds.”

May combine two claims.

Wound isolates lacked Pseudomonas virulence factors and genes for QS molecules.

“Our study has also shown the lack of Pseudomonas virulence factors and genes for QS molecules within wound isolates, which goes in line with previous studies [64] confirming that spontaneous QS- and virulence-deficient P. aeruginosa mutants are still capable of causing wound infections.”

May combine two claims.

The expression of IL-10, an anti-inflammatory cytokine, was significantly up-regulated in wounds infected with Pseudomonas, while USA300 or mixed-species infection did not cause significant induction of IL-10, although an increasing trend of IL-10 was observed in these wounds.

“The expression of IL-10, an anti-inflammatory cytokine, was significantly up-regulated in wounds infected with Pseudomonas, while USA300 or mixed-species infection did not cause significant induction of IL-10, although an increasing trend of IL-10 was observed in these wounds.”

May combine two claims.

Even though infection inhibited early epithelialization, both uninfected and infected wounds epithelialized by day 4.

“Even though infection inhibited early epithelialization, both uninfected and infected wounds epithelialized by day 4.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

S. aureus, in its methicillin sensitive and methicillin resistant form (MRSA), is a common opportunistic pathogen, responsible for the majority of all superficial skin infections, resulting in increased morbidity, mortality, and tremendous health-care costs.

“S. aureus, in its methicillin sensitive and methicillin resistant form (MRSA), is a common opportunistic pathogen, responsible for the majority of all superficial skin infections, resulting in increased morbidity, mortality, and tremendous health-care costs [20].”

• cites [20] DA Talan, 2011, Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency department patients, 2004 and 2008, Clin Infect Dis sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

USA300 appears to have enhanced virulence as compared to the traditional hospital-associated MRSA strains.

“which appears to have enhanced virulence as compared to the traditional hospital-associated MRSA strains [26], [27], [28].”

• cites [26] M Li, 2009, Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus, Proc Natl Acad Sci U S A sourceNot checked yet

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• cites [27] M Li, 2010, Comparative analysis of virulence and toxin expression of global community-associated methicillin-resistant Staphylococcus aureus strains, J Infect Dis sourceNot checked yet

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• cites [28] JM Voyich, 2005, Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils, J Immunol sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

A recent study demonstrated the ability of polymicrobial biofilms to impair healing in a rabbit ear model.

“a recent study demonstrated the ability of polymicrobial biofilms to impair healing in a rabbit ear model [39]”

• cites [39] Seth AK, Geringer MR, Galiano RD, Leung KP, Mustoe TA, <etal>et al</etal>.. (2012) Quantitative Comparison and Analysis of Species-Specific Wound Biofilm Virulence Using an In Vivo, Rabbit-Ear Model. J Am Coll Surg. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The experimental animal protocols were approved by the University of Miami Institutional Animal Care and Use Committee and all the procedures followed the federal guidelines for the care and use of laboratory animals.

“The experimental animal protocols were approved by the University of Miami Institutional Animal Care and Use Committee and all the procedures followed the federal guidelines for the care and use of laboratory animals.”

Probably not a substantial claim: may not pass S3 (load-bearing).

These virulence factors include Panton-Valentine leukocidin (pvl), staphylococcal protein A (spa) and α-hemolysin (hla).

“These virulence factors include Panton-Valentine leukocidin (pvl), staphylococcal protein A (spa) and α-hemolysin (hla) [24].”

• cites [24] M Otto, 2010, Basis of virulence in community-associated methicillin-resistant Staphylococcus aureus, Annu Rev Microbiol sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

S. aureus and P. aeruginosa are among the most common organisms isolated from both acute and chronic wounds of various etiologies.

“Staphylococcus aureus and Pseudomonas aeruginosa are among the most common organisms isolated from both acute and chronic wounds of various etiologies.”

• cites [1] Fadeev SB, Nemtseva NV (2009) [Formation of biofilms by agents of surgical soft tissue infections]. Zh Mikrobiol Epidemiol Immunobiol: 114–117.Not checked yet

  Not checked in the free run.

• cites [2] HR Champion, 2003, A profile of combat injury, J TraumaNot checked yet

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• cites [3] 2011, Prevalence of methicillin-resistant staphylococcus aureus in a combat support hospital in Iraq, Mil Med sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Increased incidence of MRSA-associated infections in both acute and chronic wounds is well-documented.

“Increased incidence of MRSA-associated infections in both acute and chronic wounds is well-documented [3], [21], [22]”

• cites [3] 2011, Prevalence of methicillin-resistant staphylococcus aureus in a combat support hospital in Iraq, Mil Med sourceNot checked yet

  Not checked in the free run.

• cites [21] MC Roghmann, 2001, MRSA colonization and the risk of MRSA bacteraemia in hospitalized patients with chronic ulcers, J Hosp Infect sourceNot checked yet

  Not checked in the free run.

• cites [22] O’Hara FP, Amrine-Madsen H, Mera RM, Brown ML, Close NM, <etal>et al</etal>.. (2012) Molecular Characterization of Staphylococcus aureus in the United States 2004–2008 Reveals the Rapid Expansion of USA300 Among Inpatients and Outpatients. Microb Drug Resist. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.

“These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.”

• AssumptionRests on an assumption: The role of bacterial virulence factors in polymicrobial wound infections has not been evaluated before this study.

Probably not a substantial claim: may not pass S3 (load-bearing).

In vitro-generated mixed species biofilms have been shown to delay healing when placed in a murine wound model.

“in vitro-generated mixed species biofilms have been shown to delay healing when placed in a murine wound model [42]”

• cites [42] T Dalton, 2011, An in vivo polymicrobial biofilm wound infection model to study interspecies interactions, PLoS One sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

KGF1 is produced during acute wound healing process mainly by fibroblasts, and has been shown to stimulate proliferation and migration of keratinocytes thus playing an important role in re-epithelialization.

“KGF1 is produced during acute wound healing process mainly by fibroblasts, and has been shown to stimulate proliferation and migration of keratinocytes thus playing an important role in re-epithelialization [53], [54].”

• cites [53] S Werner, 1992, Large induction of keratinocyte growth factor expression in the dermis during wound healing, Proc Natl Acad Sci U S A sourceNot checked yet

  Not checked in the free run.

• cites [54] I Pastar, 2008, Role of keratinocytes in healing of chronic wounds, Surg Technol IntNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Multiple studies have emphasized the presence of bacteria and the polymicrobial nature of chronic, non-healing wounds, and the frequency of S. aureus and P. aeruginosa organisms has been shown to be exceedingly high.

“Multiple studies have also emphasized the presence of bacteria and the polymicrobial nature of chronic, non-healing wounds [6], [7], [8], [9], [10], and the frequency of S. aureus and P. aeruginosa organisms has been shown to be exceedingly high [10], [11], [12], [13], [14].”

• cites [6] DN Frank, 2009, Microbial diversity in chronic open wounds, Wound Repair Regen sourceNot checked yet

  Not checked in the free run.

• cites [7] MA Pfaller, 1989, Susceptibility of nosocomial isolates of Candida species to LY121019 and other antifungal agents, Diagn Microbiol Infect Dis sourceNot checked yet

  Not checked in the free run.

• cites [8] V Gontcharova, 2010, A comparison of bacterial composition in diabetic ulcers and contralateral intact skin, Open Microbiol J sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

For each sample, a mock reaction without addition of reverse transcriptase was performed.

“For each sample, a mock reaction without addition of reverse transcriptase was performed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The leading cause of community-associated bacterial infections in the United States is the MRSA isolate USA300.

“The leading cause of community-associated bacterial infections in the United States is the MRSA isolate USA300 [25]”

• cites [25] MD King, 2006, Emergence of community-acquired methicillin-resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections, Ann Intern Med sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

The specificity of all primers was confirmed by sequencing of PCR products.

“The specificity of all primers was confirmed by sequencing of PCR products.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Toxins as well as virulence factors produced by MRSA contribute in a major way to the pathogenicity.

“toxins as well as virulence factors produced by MRSA contribute in a major way to the pathogenicity [23].”

• cites [23] G Schiavo, 2001, The bacterial toxin toolkit, Nat Rev Mol Cell Biol sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

LasI-LasR and RhlI-RhlR have been shown to regulate the expression of virulence factors such as exoenzyme S (ExoS) and exotoxin A (ToxA) which can further induce apoptosis in macrophages and neutrophils.

“LasI-LasR and RhlI-RhlR have been shown to regulate the expression of virulence factors such as exoenzyme S (ExoS) and exotoxin A (ToxA) which can further induce apoptosis in macrophages and neutrophils [33], [34], [35], [36].”

• cites [33] JP Heggers, 1992, Pseudomonas aeruginosa exotoxin A: its role in retardation of wound healing: the 1992 Lindberg Award, J Burn Care Rehabil sourceNot checked yet

  Not checked in the free run.

• cites [34] M Fazli, 2011, Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds, Wound Repair Regen sourceNot checked yet

  Not checked in the free run.

• cites [35] DG Storey, 1998, Pseudomonas aeruginosa lasR transcription correlates with the transcription of lasA, lasB, and toxA in chronic lung infections associated with cystic fibrosis, Infect Immun sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Differences between the present results and those of the rabbit ear model may be explained by variability between bacterial strains and animal models utilized.

“Our results contrast those of a recent study in a rabbit ear model where increased expression of pro-inflammatory cytokines TNFα and IL-1β was seen with polymicrobial biofilm infection [39] and these differences may be explained by the variability between bacterial strains and animal models utilized.”

May combine two claims.

Bacterial synergy can alter virulence and delay healing in multi-species wound infections.

“Our data underline the importance of bacterial interactions in multi-species wound infections as we demonstrate that bacterial synergy can alter virulence, delay healing and perhaps even change susceptibility of microbial communities to antimicrobial therapy.”

Classified as probably not a substantial claim.

Pseudomonas has two QS systems, LasI-LasR and RhlI-RhlR, shown to be important for wound infection and biofilm formation in rodent models.

“Pseudomonas has two QS systems, LasI-LasR and RhlI-RhlR, shown to be important for wound infection and biofilm formation in rodent models [32], [36].”

• cites [32] G Nakagami, 2011, Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in pressure ulcer infection in rats, Wound Repair Regen sourceNot checked yet

  Not checked in the free run.

• cites [36] KP Rumbaugh, 1999, Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections, Infect Immun sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The increased pvl and hla expression suggests an overall induction of USA300 virulence and its detrimental effects on wound healing in polymicrobial infections.

“The presence of P. aeruginosa within the polymicrobial wound environment resulted in a marked increase of pvl and hla expression suggesting an overall induction of USA300 virulence and its detrimental effects on wound healing in polymicrobial infections.”

Probably not a substantial claim: may not pass S3 (load-bearing).

pvl and spa were not important for USA300 penetration through intact epidermis.

“The same study indicated that pvl and spa were not important for USA300 penetration through intact epidermis [72], however this data cannot be fully extrapolated to the wound healing process where the epidermal barrier is compromised,”

• cites [72] G Soong, 2012, Staphylococcus aureus activation of caspase 1/calpain signaling mediates invasion through human keratinocytes, J Infect Dis sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

A recent study in a rabbit ear model found increased expression of pro-inflammatory cytokines TNFα and IL-1β with polymicrobial biofilm infection.

“Our results contrast those of a recent study in a rabbit ear model where increased expression of pro-inflammatory cytokines TNFα and IL-1β was seen with polymicrobial biofilm infection [39] and these differences may be explained by the variability between bacterial strains and animal models utilized.”

• cites [39] Seth AK, Geringer MR, Galiano RD, Leung KP, Mustoe TA, <etal>et al</etal>.. (2012) Quantitative Comparison and Analysis of Species-Specific Wound Biofilm Virulence Using an In Vivo, Rabbit-Ear Model. J Am Coll Surg. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

KGF1 is produced by fibroblasts and acts in a paracrine fashion through the KGFR2IIIb receptor found exclusively on keratinocytes, resulting in increased migration and proliferation during wound healing.

“Produced by fibroblasts, KGF1 acts in a paracrine fashion through the KGFR2IIIb receptor found exclusively on keratinocytes, resulting in increased migration and proliferation during wound healing [53].”

• cites [53] S Werner, 1992, Large induction of keratinocyte growth factor expression in the dermis during wound healing, Proc Natl Acad Sci U S A sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The polymicrobial nature of non-healing wounds, such as venous, pressure, and diabetic foot ulcers, is well documented and established.

“The polymicrobial nature of non-healing wounds, such as venous, pressure, and diabetic foot ulcers is well documented and established [6], [7], [8], [9], [58], [59].”

• cites [6] DN Frank, 2009, Microbial diversity in chronic open wounds, Wound Repair Regen sourceNot checked yet

  Not checked in the free run.

• cites [7] MA Pfaller, 1989, Susceptibility of nosocomial isolates of Candida species to LY121019 and other antifungal agents, Diagn Microbiol Infect Dis sourceNot checked yet

  Not checked in the free run.

• cites [8] V Gontcharova, 2010, A comparison of bacterial composition in diabetic ulcers and contralateral intact skin, Open Microbiol J sourceNot checked yet

  Not checked in the free run.